Accepted Manuscript Thermal denaturation kinetics of whey proteins in reverse osmosis and nanofiltration sweet whey concentrates Melanie Marx, Ulrich Kulozik PII:
S0958-6946(18)30123-7
DOI:
10.1016/j.idairyj.2018.04.009
Reference:
INDA 4311
To appear in:
International Dairy Journal
Received Date: 14 February 2018 Revised Date:
19 April 2018
Accepted Date: 23 April 2018
Please cite this article as: Marx, M., Kulozik, U., Thermal denaturation kinetics of whey proteins in reverse osmosis and nanofiltration sweet whey concentrates, International Dairy Journal (2018), doi: 10.1016/j.idairyj.2018.04.009. 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
Thermal denaturation kinetics of whey proteins in reverse osmosis and nanofiltration sweet whey concentrates Melanie Marx*,a,b and Ulrich Kulozika, b a
Technical University of Munich, Chair for Food and Bioprocess Engineering, Freising,
ZIEL Institute for Food & Health, Technical University of Munich, Weihenstephaner Berg
1, Freising, Germany
*Corresponding author: Melanie Marx
[email protected]
Tel.:
+49 8161-71 3855
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E-Mail:
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Germany
Abstract:
Shelf-stable whey concentrates produced by membrane filtration represent an energy efficient alternative to whey powder. However, to obtain products with sufficient microbiological shelf-life, preservation of concentrates is necessary. The idea of this study was, therefore, to
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investigate the influence of ionic composition and dry matter (DM) of whey concentrates on thermal stability of the major whey proteins. Taking the thermal impact of heating up into account, denaturation kinetics of β-lactoglobulin and α-lactalbumin were determined in reverse osmosis (RO) and nanofiltration (NF) whey concentrates with DM contents of 12-30%
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at heating temperatures of 90 and 125 °C. Denaturation caused by heating up was strongly increased with increasing DM of both types of concentrates. During holding at 90 °C, whey proteins showed a higher thermal stability in RO concentrates as compared to NF concen-
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trates. However, denaturation rates at 125 °C of whey proteins were lower in NF than in RO concentrates.
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1. Introduction Whey proteins are important additives for various food applications because of their techno-
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functional properties and their high content of essential amino acids. Evaporation and subse-
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quent spray drying are still the methods of choice to obtain whey proteins in a stable form.
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However, spray drying is the most energy intensive process step in dairy industry. The solu-
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bility of α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) is, furthermore, strongly affected at
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high outlet gas temperatures (100-120°C) during spray drying (Anandharamakrishnan, Rielly,
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& Stapley, 2008).
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Using energy-efficient membrane filtration performed at cold temperatures (10 °C), the whey
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proteins are kept in the native state in contrast to evaporation performed at much higher tem-
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peratures (60-71 °C) (Jelen; Plock, 1994). The composition of the concentrate is determined
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by the retention behavior of the used membrane and, thus, can be adapted to its further appli-
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cation. By means of reverse osmosis (RO), all solutes of the whey are retained by the mem-
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brane. Therefore, RO whey concentrates and concentrates produced by evaporation show al-
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most the same composition. Using nanofiltration (NF), however, concentration and partial
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demineralisation of whey can be combined in one process. A reduction in ash content of 40-
17
60% is obtainable for a concentration factor of 4-5 during NF of whey (Gernigon, Schuck,
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Jeantet, & Burling, 2011).
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By skipping the energy-intensive spray-drying step after concentration, huge amounts of en-
20
ergy could additionally be saved. In contrast to whey powder, the water activity of whey con-
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centrate, however, is too high to prevent microbial growth. Therefore, thermal treatment of
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concentrates is necessary to guarantee sufficient shelf-life.
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To keep whey protein denaturation during heat treatment of whey concentrates as low as pos-
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sible, it is necessary to determine the denaturation rate of the major whey proteins in the re-
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spective heating medium. The thermal denaturation of α-La and β-Lg is a complex process
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described in detail by De Wit (2009), Tolkach & Kulozik (2007), Havea, Singh, & Creamer
27
(2000), Permyakov & Berliner (2000) and Schokker, Singh, & Creamer (2000).
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In previous studies, the thermal denaturation behavior of whey proteins in whey, whey con-
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centrated by evaporation, and whey protein solutions has been investigated (Dickow, Kauf-
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mann, Wiking, & Hammershøj, 2012; Tolkach & Kulozik, 2007; Plock, Spiegel, & Kessler,
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1997; Dannenberg, 1986). However, based on these studies, it is not possible to predict
32
thermal denaturation behavior of whey proteins in whey concentrated by RO or NF up to 30%
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DM. On the one hand, whey concentrated by membrane filtration contains a higher concentra-
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tion of native whey proteins than whey concentrated by evaporation which influences thermal
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ACCEPTED MANUSCRIPT stability. On the other hand, nature and concentration of solutes in the heating medium greatly
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influence the thermal stability of whey proteins.
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Lactose accounts for about 75% of the dry matter of whey and whey concentrates and is
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known to stabilize protein structures (Shimizu & Smith, 2004). However, the stabilizing ef-
39
fect of lactose on α-La is strongly dependent on the type of the heating medium (Anema, Lee,
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& Klostermeyer, 2006; Plock, Spiegel, & Kessler, 1998). Another factor affecting whey
41
protein denaturation is the protein concentration itself; the higher the concentration of whey
42
proteins, the higher the reaction rate for the denaturation (Wolz & Kulozik, 2015; Verheul,
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Roefs, & Kruif, 1998). By concentrating whey using RO or NF, the concentration of whey
44
proteins in whey increases from 0.7% to 4% in whey concentrates with 30% DM. Further-
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more, the salt concentration of whey concentrates represents an important factor on whey pro-
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tein denaturation. The effect influencing the thermal stability of whey proteins is also deter-
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mined by the nature of the ionic components. Using NF, the concentration of monovalent ions
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in the whey concentrates decreases strongly, whereas losses of multivalent ions and lactose
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are negligible. Calcium is generally known to accelerate the heat-induced aggregation of β-Lg
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(Petit, Herbig, Moreau, & Delaplace, 2011; Simons et al., 2002; Visser, 1992), but high con-
51
centrations of calcium can also result in a deceleration of protein unfolding (Visser, 1992).
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Sodium chloride increases the denaturation temperature of β-Lg with increasing concentration
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(Nicorescu et al., 2008). Different ions in variable concentrations influence the shielding ef-
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fect of electric charges of proteins in general which vary as a function of pH. Antagonistic
55
interactions between the different salts and lactose are expected to affect the protein stability
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in whey concentrates. The lower ionic strength of NF concentrates compared to RO concen-
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trates might have a positive effect on the thermal stability of whey proteins. The hypothesis,
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therefore, is that the thermal stability of whey proteins in whey, RO and NF whey concen-
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trates varies because of the differences in their low molecular solute composition.
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In addition to the composition of the heating medium, the reaction rate of the denaturation
61
reaction is strongly influenced by the applied process conditions. The treatment temperature
62
has a strong effect on the denaturation reaction of the whey proteins. With increasing tem-
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perature, the reaction rate increases, whereby the rate is determined by the unfolding reaction
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at lower temperatures (≤ 90 °C) and by the aggregation reaction at higher temperatures
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(˃ 90 °C) (Tolkach & Kulozik, 2007; Anema et al., 2006; Schokker, Singh, & Creamer, 2000;
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Plock et al., 1997).
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ACCEPTED MANUSCRIPT Thus, depending on the heating temperature and rate as well as the composition of the medi-
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um, the heating-up phase to the desired temperature can already lead to thermal denaturation
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of whey proteins.
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In previous studies, in which also the reaction kinetics of whey protein denaturation were in-
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vestigated by means of the same heating system as in this study, the thermal impact of the
72
heating-up phase was not considered. This means that the heat-holding phase starts when the
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heating-up phase of the samples is started (Leeb, Haller, & Kulozik, 2018; Spiegel & Huss,
74
2002). However, the heating media in these studies were protein solutions (protein concentra-
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tion 5-10%) without salts or with only low ionic strength. At these protein concentrations,
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proportions of denatured proteins after the heating-up phase are negligible. Due to the high
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concentration of solutes and the high ionic strength of whey concentrates, thermal denatura-
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tion of whey proteins is, however, expected to be enhanced and might occur to a considerable
79
extent during the phase of heating-up to high temperatures (> 100 °C).
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Therefore, we decided to follow the recommendation of Whitaker (2003) and take the heat-
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ing-up phase into account by immediate cooling of the samples after the heating-up phase.
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The remaining concentration of native protein after heating up was used as reference value for
83
the calculation of the reaction kinetics.
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The aim of this study was, therefore, to investigate the influence of the ionic composition and
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the DM content of whey concentrates on thermal denaturation kinetics of whey proteins, con-
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sidering the thermal impact of the heating-up phase.
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2. Materials & Methods
2.1 Preparation of RO and NF whey concentrates with defined dry matter contents
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Sweet whey from hard cheese production was obtained from the dairy MEGGLE Wasserburg
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GmbH & Co. KG (Wasserburg, Germany). Table 1 shows the composition of the sweet whey.
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Sweet whey was concentrated by means of RO or NF up to a DM content of 30% on a pilot
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scale at a temperature of 10 °C and transmembrane pressure of 40 bar (RO) or 20-30 bar
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(NF). Permeates of RO and NF were collected and used for redilution of the retentates to de-
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fined DM contents of 12-30%. The salt retention of the used RO membranes was > 99.2%
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based on standard testing conditions (FILMTEC™ SW30-2540, FilmTec Corporation, Edina,
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MN, USA, and Koch TFC®-SW 2.5”, Koch Membrane Systems, Aachen, Germany). Table 1
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summarizes the composition of the RO whey concentrates.
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The salt rejection of the used NF membrane of 98% was slightly lower as compared to the RO
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membrane (GE DK Series Thin Film Membrane, General Electric Company, Fairfield, CT,
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USA). The composition of the NF whey concentrates is depicted in Table 2. 3
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addition of 1 M or 0.1 M NaOH or HCl with vigorous stirring.
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2.2 Analytical Methods
104
Lactose concentration of feed, permeate and retentate from RO and NF whey concentrates
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was analyzed by high-performance liquid chromatography using an Aminex HPX-87H
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(300 x 7.8 mm) column (Bio-Rad Laboratories GmbH, München, Germany) and an Agilent
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1100 chromatograph (Santa Clara, CA, USA). Using a nitrogene analyser vario MAX cube,
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the total protein content was measured by determination of the total nitrogen concentration
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with conversion to protein content (Elementar Analysensysteme GmbH, Langenselbold,
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Germany) according to the method of Dumas. For determining the concentration of calcium,
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sodium and potassium flame photometry was performed by means of a flame photometer
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ELEX 6361 (Eppendorf AG, Hamburg, Germany). The total dry matter content was measured
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by differential weighing using a smart turbo micro wave dryer (CEM GmbH, Kamp-Lintfort,
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Germany). To determine and adjust the pH-value of the solutions, a pH meter (Xylem Analyt-
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ics Germany Sales GmbH & Co. KG, Weilheim, Germany) was used.
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2.3 Thermal treatment of whey and whey concentrates
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Four mL of whey or whey concentrates were filled in stainless steel tubes for thermal treat-
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ment. Heat treatment was performed by means of a water bath (WB 22, Memmert
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GmbH + Co. KG, Schwabach, Germany) at heating temperatures < 100 °C. After the thermal
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treatment, the samples were put into ice water for fast cooling. A pressure vessel heated by
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saturated steam was used for treatment temperatures > 100 °C. Using cold tap water, the sam-
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ples were cooled. In the study of Behringer (1989), the pressure vessel is explained in detail.
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By use of a data logger connected to a temperature sensor in a reference tube, the temperature
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profile of the thermal treatment was recorded. Marx & Kulozik (2018) presented a typical
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temperature profile of heat treatment in the pressure vessel at a temperature of 125 °C. When
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the desired treatment temperature, e.g., 125 °C, was reached, the heat holding phase started.
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To subtract the effect of the heating phase, the heat treatments were also performed without
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holding phase. In this case, the samples were immediately cooled down after reaching the
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desired treatment temperature. This treatment was used as zero point whose concentration is
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defined as c0 as proposed by Whitaker (2003). All heat treatment experiments were conducted
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at least in triplicate.
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The concentrations of native α-La and β-Lg were determined by means of reversed phase-
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high performance liquid chromatography (RP-HPLC) as described by Toro-Sierra, Tolkach,
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& Kulozik (2013). To evaluate the effect of the heating-up phase on the nativity of the whey
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proteins, the concentrations of native protein were measured in the untreated sample cuntreated
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and after the heating-up phase cheat up.
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For the calculation of reaction kinetics of thermal denaturation of whey proteins, the protein
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concentration after defined heat-holding times ct was additionally measured. As already men-
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tioned, the concentration of native whey proteins after heating up cheat up was defined as initial
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concentration c0 for calculation of reaction kinetics in Section 2.5.
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The contents of native α-La and β-Lg were determined after the denatured proteins were pre-
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cipitated at pH 4.6 and separated using a syringe filter with a cut-off of 0.45 µm, while the
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native whey proteins remained soluble and permeated through the filter.
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The degree of denaturation (DD) after heating up was calculated by equation (1):
= 1−
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× 100%
(1)
where Cuntreated is the initial protein concentration and Ct is the concentration of native β-Lg
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after a heating time, t.
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2.5 Evaluation of reaction kinetics
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The changes in the native protein concentration caused by thermal treatment can be mathe-
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matically described. The reaction rate of the denaturation reaction is determined by the reac-
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tion order, n, the native protein concentration, c, and the temperature-dependent rate constant,
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kT/n, and can be described by equation (2):
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−
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For a reaction order of n ≠ 1.0 the integration of equation (2) results in equation (3):
=
=
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.
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/
× ! × "# − 1$ ×
(2)
%
"
$
+ 1
(3)
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and for n = 1.0, equation (4) is obtained by integrating equation (2):
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ln
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The reaction order of the denaturation reaction of α-La and β-Lg was adjusted to a value re-
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sulting in a fit of the data points by straight lines according to the linear equations (2) or (3).
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The rate constants, kT/n, of the thermal denaturation of α-La and β-Lg at the different tempera-
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tures were calculated from the slopes of the regression lines.
= −
/
× !
(4)
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The temperature dependence of the concentration independent reaction constant kT/n can be
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described by the Arrhenius equation:
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=
/
%/
× '
()* +×,
(5)
where k0/n is the pre-exponential factor, Ea is activation energy, R is the universal gas constant
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(8.314 J mol-1 K-1) and T is absolute temperature.
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2.6 Statistical analysis
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All experiments were at least performed in triplicate. Linear regression of the protein denatur-
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ation data was performed using OriginPro 2015 (OriginLab Corporation, Northampton, MA,
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USA) for Windows. All data points in figures show mean values ± standard deviation calcu-
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lated by use of the Student’s t-distribution. The temperature dependent rate constants kT/n
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(Tables 4 and 5) are given with a 95% confidence interval calculated from the regression
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lines.
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3. Results and Discussion
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In the present study, the denaturation kinetics of the major whey proteins α-La and β-Lg in
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RO and NF whey concentrates were investigated. Since environmental conditions are known
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to affect the thermal stability of whey proteins, the impact of the DM content (12-30% DM)
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and the mineral composition of whey concentrates on heat stability was studied during heat
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treatment at 90 °C and 125 °C. There is a change in temperature dependence of the denatura-
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tion reaction at temperatures close to 95 °C observed in milk and cheese whey (Hillier & Lys-
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ter, 1979). Thus, the influence of both temperature ranges was studied. Variations of the min-
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eral composition were achieved by concentration of whey by means of NF. By concentrating
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whey, the water content is reduced not only, but also the pH-value. During the concentration
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of whey by RO from 6 to 30% DM, the pH decreases from 6.5 to about 5.9. The effect of the
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NF on the pH is similar, but a little less pronounced. Thermal resistance of the whey proteins
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is strongly affected by the pH of the heating medium (O'Kennedy & Mounsey, 2009; Leeb et
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al., 2018). To avoid interactive effects, we adjusted the pH of all concentrates to the initial pH
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of the whey (pH 6.5) prior to the heat treatment.
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The integral method was used to identify the reaction order of the denaturation reaction of α-
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La and β-Lg in whey and the differently composed whey concentrates. When the results are
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plotted according to equation (3) or (4) with reaction order n as a variable, n is done such that
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the data fulfill the requirement of a linear equation yielding a straight line in the data plot. For
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the range of investigated denaturation temperatures, a reaction order for β-Lg of n=1.5 was
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agreement with the results of previous studies carried out in concentrated whey, skim milk
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and whey protein solutions at neutral pH (Leeb et al., 2018; Wolz & Kulozik, 2015; Tolkach
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& Kulozik, 2007; Anema et al., 2006; Plock et al., 1998). The denaturation reaction of α-La
198
was, however, best described by a reaction of the order of n=1.0. This is in accordance with
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the results of Anema & McKenna (1996) and Dannenberg & Kessler (1988), who investigated
200
the reaction kinetics of whey proteins in milk. In addition, the thermal impact of the heating-
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up phase was assessed by immediate cooling after the heat-holding temperature was reached.
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The remaining concentration of native whey protein was used as reference value for determi-
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nation of reaction kinetics.
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3.1 Thermal denaturation of whey proteins during heating-up phase
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Thermal denaturation of whey proteins starts already during the heating-up phase to the
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desired heat-holding temperature. Therefore, we determined the degrees of denaturation (DD)
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of α-La and β-Lg after heating up to 90 and 125 °C (Fig. 1). As shown in Fig. 1 A, the DD of
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β-Lg after heating up to 90 °C is dependent on the DM content and the ionic composition of
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the concentrates. In RO concentrates, an increase of the DM from 6 to 18% results in an in-
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crease of the DD from 40 to 50%, whereas further increase of the DM up to 30% leads to in-
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creased thermal stability of β-Lg, which might be due to a protective effect of the non-protein
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soluble components, which Anema et al. (2006) also observed in milk.
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However, by increasing the DM from 12 to 30% in NF whey concentrates, the DD of β-Lg is
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increasing by 15% in total. Thus, no protective effect of the increased DM content can be ob-
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served in NF concentrates, which might be explained by the reduced concentration of NaCl in
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NF concentrates (Suárez, Lobo, Alvarez, Riera, & Álvarez, 2009; Suárez, Lobo, Álvarez,
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Riera, & Álvarez, 2006).
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By heating up to 125 °C (Fig. 1 C), the DD of β-Lg rises from 75% to 95% with increasing
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the DM from 6 to 30% independent of the used membrane method. We explain this accelera-
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tion of the thermal denaturation of β-Lg at 125 °C by the increased initial protein content of
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concentrates with higher DM contents on the one hand and the reduced stabilizing effect of
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lactose at temperatures > 110 °C (Anema et al., 2006).
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Table 3 shows the residual native protein concentration of β-Lg after the heating-up phase to
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125 °C without heat holding in whey, RO and NF whey concentrates. The remaining native
225
protein concentration of RO concentrates is decreasing with increasing DM content from 12
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to 30%, although the RO whey concentrate with 30% DM content contains the highest initial
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protein concentration before heating (Tables 1 and 2). In NF concentrates, the native protein 7
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ACCEPTED MANUSCRIPT concentration is increasing with increasing DM content from 12 to 24%. Compared to the NF
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concentrates with 12-24% DM, only the NF concentrate with 30% DM shows a significantly
230
reduced remaining protein concentration, which is similar to that of whey. The accelerated
231
denaturation reaction of β-Lg in NF concentrate with 30% DM might be explained by the
232
higher initial protein content and the reduced concentration of monovalent ions as also de-
233
scribed for a heating temperature of 90 °C. Since the initial protein concentration of whey is
234
lower than the protein concentration of the concentrates, β-Lg shows the lowest DD at 125 °C
235
after the heating-up phase in whey.
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As can be seen from Figs. 1 B and D, α-La showed a higher thermal stability than β-Lg at
237
both heating temperatures. By heating up to 90 °C, only 10% of α-La are denatured in whey
238
as well as RO and NF whey concentrates with 12% DM. With increasing DM from 18 to
239
30%, the DD increases by about 10% for RO and NF whey concentrates. The thermal stability
240
of α-La is slightly higher in RO than in NF concentrates which show the highest DD of 22%
241
in NF concentrate with 30% DM. During the heating-up phase to 125 °C, the influence of the
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DM as well as of the composition of the concentrate on the thermal stability of α-La is in-
243
creased as compared to 90 °C. The lowest DD of 24% was measured in whey. In RO and NF
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whey concentrates, the DD of α-La was significantly increased for higher DM contents and
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reached a maximum value of 64% in RO concentrate with 30% DM. In NF concentrates, the
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DD showed a linear dependence on the DM content, which was slightly less pronounced than
247
in RO concentrates. The remaining concentration of native α-La after heating up to 90 or
248
125 °C is shown in Table 3. In contrast to β-Lg, the remaining concentration of native α-La
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increased with increasing DM contents in all RO and NF concentrates analyzed.
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Based on the remaining protein concentrations after heating up, reaction kinetics for the dena-
251
turation of whey proteins during heat-holding at 90 or 125 °C were calculated. Since the main
252
objective of the study was to compare RO and NF concentrates with different DM contents,
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we also determined the reaction kinetics for the concentrates showing already high DD after
254
heating up to 125 °C.
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3.2 Reaction kinetics of β-Lg in whey and RO whey concentrates
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Figs. 2 A and B show the denaturation of β-Lg in sweet whey as a control, i.e., prior to the
257
concentration, and RO whey concentrates (12-30% DM) at a heating temperature of 90 and
258
125 °C plotted as a reaction of the order of n=1.5 in dependence of the heat-holding time,
259
which starts when the desired heating temperature is reached in the respective sample. At a
260
heating temperature of 90 °C, β-Lg showed a similar denaturation behavior in whey and RO
261
concentrates with 12, 18 and 30% DM and in whey, whereas an increase of the DM from 18 8
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ACCEPTED MANUSCRIPT to 24% leads to increased denaturation. As several studies have shown, the denaturation reac-
263
tion of β-Lg is greatly dependent on the environmental conditions of the heating medium,
264
especially in the unfolding limited temperature area (≤ 90 °C) (Dissanayake, Ramchandran,
265
Donkor, & Vasiljevic, 2013; Erabit, Flick, & Alvarez, 2013). When β-Lg is heated in pure
266
protein solutions, an increase of the protein concentration results in an acceleration of the de-
267
naturation reaction (Anema et al., 2006; Wolz & Kulozik, 2015). However, in complex solu-
268
tions, such as whey or whey concentrates, the non-protein soluble components, which account
269
for 85-90% of the total DM content (Tables 1, 2 and 3), also influence the denaturation behav-
270
ior of β-Lg to a great extent. Lactose has a protective effect on the whey proteins that can
271
overlay the negative effect of increasing native protein concentration with increasing dry mat-
272
ter (Plock et al., 1998; Anema et al., 2006). Depending on their nature and concentration, salts
273
can either promote or decelerate whey protein denaturation. However, by using RO for con-
274
centration of whey, the ratio between protein content and the content of non-protein soluble
275
components remains constant during concentration. It is assumed that this protective effect of
276
the non-protein dry matter, especially the lactose, is the reason for the similar denaturation
277
rates of β-Lg in RO whey concentrates (12, 18, 30% DM) and whey at 90 °C. However, β-Lg
278
shows a decreased thermal stability in RO whey concentrate with 24% DM.
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By increasing temperature from 90 to 125 °C, the denaturation reaction of β-Lg is accelerated
280
according to the Arrhenius equation (equation (5)) which becomes apparent by the steeper
281
slopes of the regression lines (Fig. 2 B). Furthermore, the impact of the DM content on the
282
denaturation reaction of β-Lg changes by the temperature increase. As can be seen from Fig. 2
283
B, higher DM contents seem to lead to retarded denaturation of β-Lg at 125 °C, except for
284
whey. Knowing that the amount of native β-Lg at the beginning of the heat holding time (0 s)
285
(Table 3) decreases with increasing DM content of the RO concentrates due to strong dena-
286
turation during heating up (Fig. 1 C), it becomes clear that the native protein concentration is
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the main influencing factor for the different slopes of the regression lines in Fig. 2 B. Due to
288
its low initial protein concentration, the protein concentration of whey was very similar to that
289
of RO concentrates with 24 and 30% DM after heating up. The number of collisions per time
290
unit is responsible for the extent of denaturation in the aggregation limited temperature area.
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3.3 Reaction kinetics of β-Lg in NF whey concentrates
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Figs. 3 A and B show the denaturation of β-Lg in NF whey concentrates (12-30% DM) at a
293
heating temperature of 90 and 125 °C plotted as a reaction of the order of n=1.5. Changes in
294
the DM content of NF concentrates have a stronger impact on the denaturation of β-Lg as
295
compared to RO concentrates at a heating temperature of 90 °C. By increasing the dry matter 9
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297
significantly (P < 0.05). However, when the DM content was further increased, we detected
298
the lowest level of β-Lg denaturation in NF concentrates with 30% DM at 90 °C (Fig. 3 A).
299
Since the denaturation of β-Lg is greatly influenced by the composition of the heating medi-
300
um at temperatures ≤ 90 °C as described for RO concentrates, we suppose that the complex
301
and changing composition of protein, lactose and salt concentration in the NF whey concen-
302
trates is responsible for the lower heat stability of β-Lg. Compared with RO whey concen-
303
trates with the same DM contents, NF whey concentrates are characterised by a reduced con-
304
centration of monovalent ions and, consequently, show slightly higher lactose and protein
305
concentrations (Tables 2 and 3). Depending on the DM content, the concentration of the mon-
306
ovalent anion chloride is reduced by 10-46% (data not shown) and the monovalent cations
307
sodium and potassium are reduced by 33-48%. In contrast, the reduction in multivalent cati-
308
ons, e.g., calcium, as a result of concentration by NF is negligible. Consequently, the ratio of
309
protein to lactose to the different salts is changing during concentration by means of NF. The
310
findings in literature show that the influence of the ionic strength on the denaturation behavior
311
of β-Lg is complex, because it is strongly dependent on the nature and the concentration of
312
the present ions (O'Kennedy & Mounsey, 2009; Simons et al., 2002; Rham & Chanton, 1984;
313
Petit et al., 2011). O'Kennedy & Mounsey (2009) observed that the level of β-Lg denaturation
314
and aggregation in a 1% (w/w) β-Lg solution was promoted by increasing concentrations of
315
calcium ions, while an increase of the ionic strength by use of NaCl overlaid this effect. How-
316
ever, Plock (1994) found a stabilizing effect of calcium at concentrations between 0.3 mg mL-
317
1
318
again to accelerated denaturation. We suppose that the complex interaction of ionic strength,
319
lactose and protein concentration in NF whey concentrates results in a differing thermal sta-
320
bility of β-Lg in the concentrates with increasing DM from 18 to 30%.
321
At a treatment temperature of 125 °C the DM also significantly affects thermal stability of β-
322
Lg (Fig. 3 B). β-Lg shows the highest thermal stability in NF whey concentrate with a DM
323
content of 30%. Minimal thermal stability of β-Lg was observed for NF whey concentrates
324
with 18 and 24% DM content. As shown in Fig. 1 C, a high proportion of β-Lg in NF whey
325
concentrates undergoes thermal denaturation during the heating-up phase to 125 °C. Except
326
for 24% DM (P < 0.05), RO and NF whey concentrates show the same DD after the heating-
327
up phase (Fig. 1 C). Thus, the remaining native concentration of β-Lg in RO and NF whey
328
concentrates after heating up only differs significantly in concentrates with 24% DM (Table
329
3). NF concentrates with 18% and 24% DM show the highest concentrations of native β-Lg at
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and 2-3 mg mL-1 in evaporated whey. Further increases in the calcium concentration led
10
ACCEPTED MANUSCRIPT the beginning of the heat-holding phase followed by 12% and 30% DM content. As already
331
described for RO concentrates, it is assumed that the concentration of native β-Lg after the
332
heating-up phase determines the denaturation rate during the heat-holding phase at 125 °C.
333
The effect of the protein concentration seems to be more important than the effect of the com-
334
position of the heating medium at 125 °C, which is in accordance with the results of Plock
335
(1994) and Dissanayake et al. (2013).
336
3.4 Temperature-dependent rate constants of the denaturation reaction of β-Lg in whey, RO
337
and NF whey concentrates at 90 °C and 125 °C
338
The temperature-dependent reaction rate constants kT/1.5 for the thermal denaturation of β-Lg
339
were calculated from the regression lines shown in Figs. 2 and 3. To illustrate the effect of the
340
DM content and of the composition of whey concentrates on the thermal denaturation of β-
341
Lg, the temperature-dependent reaction rate constants for β-Lg in whey, RO and NF whey
342
concentrates (12-30 % DM) are summarized in Table 4.
343
At 90 °C the lowest reaction rate constants were found for NF whey concentrate with 30%
344
DM (11.2 x 10-3 s-1), whey (11.7 x 10-3 s-1) and RO whey concentrate with 12% DM
345
(12.6 x 10-3 s-1), which were not statistically different (P ≥ 0.05). In NF whey concentrate with
346
24% DM, we found the highest reaction rate constant with 30.4 x 10-3 s-1. In both types of
347
concentrates, the highest rate constants were found for a DM of 24% at a heating temperature
348
of 90 °C. Comparing concentrates with the same DM content, it becomes clear that thermal
349
denaturation of β-Lg is accelerated in NF concentrates for 12, 18 and 24% DM. Leeb et al.
350
(2018) determined a temperature dependent rate constant of 31.4 x 10-3 s-1 for a 5% β-Lg so-
351
lution at pH 6.8 and a heating temperature of 90 °C, which is in good agreement with our re-
352
sults for NF whey concentrate with 24% DM, which had a total protein content of 3.16%.
353
At 125 °C, the lowest reaction rate constants of 33.7 x 10-3 s-1 were again found for NF whey
354
concentrate with 30% DM. We determined the highest reaction rate constants in RO whey
355
concentrates with 12 (98.9 x 10-3 s-1) and 18% DM (85.6 x 10-3 s-1), which were not statistical-
356
ly different (P ≥ 0.05). In contrast to the results obtained at a treatment temperature of 90 °C,
357
the rate constants in RO concentrates tend to be higher than in NF whey concentrates for the
358
denaturation of β-Lg at 125 °C. In our opinion, this difference in the thermal stability of β-Lg
359
could be caused by the reduced salt concentration of NF concentrates as compared to RO con-
360
centrates. Our findings are of the same order of magnitude as the results of Tolkach
361
& Kulozik (2007), who determined a rate constant of 45 x 10-3 s-1 for a 5% β-Lg solution at
362
neutral pH and at a temperature of 120 °C.
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ACCEPTED MANUSCRIPT 3.5 Reaction kinetics of α-La in whey and RO whey concentrates
365
Fig. 4 shows the time dependent denaturation behavior of α-La in whey and RO whey concen-
366
trates (12-30% DM) at 90 °C and 125 °C as a reaction of the order of n=1. The denaturation
367
rates of α-La are very similar for the different DM at 90 °C. Only in RO whey concentrate
368
with 12% DM and whey, α-La showed a slightly increased thermal stability.
369
The denaturation rate of α-La was significantly accelerated at 125 °C compared to 90 °C. In
370
contrast to β-Lg, α-La showed a higher thermal stability and, therefore, much lower degrees
371
of denaturation after the heating-up phase. Thermal stability of α-La during heat-holding at
372
125°C was only marginally influenced by the DM of the whey concentrates. Solely for con-
373
centrate with 30% DM, the denaturation of α-La was slightly increased compared to concen-
374
trate with 12, 18 or 24% DM and whey, although the initial native protein concentration at the
375
beginning of the heat-holding time continuously increased with increasing DM (Table 3).
376
Anema & McKenna (1996) also reported that the rate constants for the denaturation of α-La
377
in reconstituted whole milk were essentially independent of the protein concentration for tem-
378
peratures > 85 °C.
379
3.6 Reaction kinetics of α-La in NF whey concentrates
380
The denaturation of α-La in NF whey concentrates (12-30% DM) at 90 °C (A) and 125 °C (B)
381
is depicted in Fig. 5. At 90 °C, we found the highest thermal stability of α-La in NF whey
382
concentrates with 30% DM, followed by NF whey concentrate with 12% DM. The fastest
383
denaturation reaction characterized by the steepest slope was determined for NF whey con-
384
centrate with 24% DM. An increase of the temperature to 125 °C results in significantly
385
steeper slopes of the regression lines. From Fig. 5 B it is evident that the denaturation reaction
386
is dependent on the DM content of the NF whey concentrates. α-La shows the same denatura-
387
tion rate for whey concentrates with 12 and 30% DM. However, the denaturation rate was
388
increased for NF concentrates with 18 and 24% DM.
389
As presented in Figs. 4 A and B, the denaturation of β-Lg showed a similar dependence on the
390
DM content of NF whey concentrates as α-La at 90 °C and 125 °C. It is known from literature
391
that the denaturation of α-La is strongly influenced by the presence of β-Lg and the availabil-
392
ity of free and reactive thiol groups. Increasing concentrations of unfolded β-Lg molecules
393
facilitate thiol disulfide interchange reactions between α-La and β-Lg and result in increased
394
levels of irreversible denaturation and aggregation of α-La. Furthermore, the degree of revers-
395
ibility of the denaturation reaction of α-La depends on the ionic environment of the solution
396
(McGuffey, Otter, van Zanten, & Foegeding, 2007). Thus, the differences in α-La denatura-
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ACCEPTED MANUSCRIPT tion rates in NF whey concentrates are probably a consequence of the denaturation of β-Lg,
398
which is greatly influenced by the varying solute concentrations at the different DM contents.
399
3.7 Temperature-dependent rate constants of the denaturation reaction of α-La in whey, RO
400
and NF whey concentrates at 90 °C and 125 °C
401
The temperature dependent denaturation rate constants kT/1.0 for the thermal denaturation of α-
402
La were calculated from the regression lines in Figs. 4 and 5. The reaction rate constants for
403
the thermal denaturation of α-La in whey, RO and NF whey concentrates (12-30 % DM) are
404
presented in Table 5. The lowest reaction rate constants for the denaturation of α-La were
405
found for whey with 3.1 x 10-3 s-1 and for RO whey concentrate with 12% DM with 3.2 x 10 -
406
3 -1
407
(1988) determined for the denaturation of α-La in milk at 90 °C.
408
In RO whey concentrates with 18, 24 and 30% DM, as well as in NF whey concentrates with
409
12 and 30% DM, the denaturation rates were not significantly different and ranged between
410
4.3 x 10-3 and 4.9 x 10-3 s-1. NF whey concentrate with 24% DM showed the highest denatura-
411
tion rate of 8.4 x 10-3 s-1 for α-La at 90 °C.
412
At a heating temperature of 125 °C, we found the lowest reaction rate constant again for
413
whey, with 13.2 x 10-3 s-1 which is in good agreement with the rate constant, which Dannen-
414
berg & Kessler (1988) determined for α-La in milk at 120 °C. In RO and NF whey concen-
415
trates, the reaction rate constants for the denaturation of α-La were significantly higher than in
416
whey. The rate constants in RO whey concentrates, that range between 41.0 x 10-3 and
417
53.8 x 10-3 s-1, increased compared to the rate constants in NF whey concentrates, which
418
ranged between 20.3 x 10-3 and 32.6 x 10-3 s-1.
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at 90 °C. These findings are in agreement with the results of Dannenberg & Kessler
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4. Conclusions
The findings of this study provide new insights in the denaturation behavior of the major
421
whey proteins α-La and β-Lg in RO and NF whey concentrates with DM contents of 12-30%.
422
It could be shown that the method used for membrane concentration as well as the DM con-
423
tent have a significant influence on the whey protein denaturation which is dependent on the
424
heating temperature. A positive effect of demineralisation by NF on whey protein stability
425
could be determined for heat treatment at 125 °C. The influence of increasing DM contents on
426
the whey protein denaturation was particularly pronounced during the heating-up phase.
427
Based on the obtained results, it is possible to control the denaturation behavior of α-La and
428
β-Lg in complex whey based solutions.
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ACCEPTED MANUSCRIPT However, especially β-Lg already shows high DD after heating up to the desired temperature.
430
For industrial applications, where very high proportions of native whey proteins are desired, it
431
therefore appears to be necessary to develop a gentler preservation process for fluid whey
432
concentrate. To produce tailor-made whey concentrates showing the desired shelf-life as well
433
as native whey protein, a two-step preservation process consisting of cold microfiltration and
434
gentle heat treatment could be more suitable in terms of native whey protein. This combined
435
preservation process and its suitability for whey concentrates will therefore be investigated in
436
further studies.
437
Acknowledgement
438
The authors gratefully thank Theodora Brodbeck, Corinna Karl, Franziska Kurz, Melanie
439
Groß, Julia Hintermayer, Anna Müller and Isabella Tallarita for experimental support. This
440
research project was supported by funds of the Federal Ministry of Food and Agriculture
441
(BMEL) based on a decision of the Parliament of the Federal Republic of Germany via the
442
Federal Office for Agriculture and Food (BLE) under the innovation support program (grant
443
number 313.06.01-28-1-74.005-11).
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ACCEPTED MANUSCRIPT Table 1.
546
Composition of whey and reverse osmosis whey concentrates with dry matter contents of 12,
547
18, 24 and 30%. Data represent mean values ± standard deviations of at least three repli-
548
cates. a
550 551 552
549 Item Whey RO 12 RO 18 RO 24 RO 30 DM, % 5.72 ± 0.12a 12.17 ± 0.24b 18.11 ± 0.18c 24.08 ± 0.13d 30.09 ± 0.31e Total protein, % 0.73 ± 0.08a 1.50 ± 0.09b 2.20 ± 0.14c 2.96 ± 0.12d 3.75 ± 0.11e thereof: α-La, g L-1 0.78 ± 0.04a 1.55 ± 0.14b 2.28 ± 0.15c 3.22 ± 0.26d 4.06 ± 0.27e β-Lg, g L-1 3.27 ± 0.30a 6.53 ± 0.59b 9.33 ± 0.39c 13.04 ± 0.49d 16.46 ± 0.95e -1 Lactose, g L 39.45 ± 2.52a 81.91 ± 5.86b 120.89± 2.86c 162.43 ± 3.24d 220.27± 4.94e Na+, g L-1 0.45 ± 0.08a 0.91 ± 0.17a,b 1.32 ± 0.16b,c 1.60 ± 0.03c 2.07 ± 0.18c K+, g L-1 1.52 ± 0.07a 2.86 ± 0.02b 4.14 ± 0.32c 5.82 ± 0.08d 6.86 ± 0.47e Ca2+, g L-1 0.40 ± 0.01a 0.78 ± 0.02b 1.15 ± 0.05c 1.57 ± 0.02d 1.94 ± 0.04e a Abbreviations are: RO, reverse osmosis whey concentrate at 12%, 18%, 24% and 30% dry matter content; DM, dry matter; Na+, sodium; K+, potassium; Ca2+, calcium. Measured values with the same superscript in one row are not statistically different (P ≥ 0.05).
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Table 2.
555
Composition of nanofiltration whey concentrates with dry matter contents of 12, 18, 24 and
556
30%. Data represent mean values ± standard deviations of at least three replicates. a
1.56 ± 0.09a 6.67 ± 0.37a 84.28 ± 4.84a 0.61 ± 0.05a 2.19 ± 0.02a 0.77 ± 0.03a
3.32 ± 0.23c 14.47 ± 0.79c 184.85 ± 2.46c 0.85 ± 0.07a 2.86 ± 0.078b 1.46 ± 0.04c
2.35 ± 0.08b 9.91 ± 0.31b 131.57 ± 11.12b 0.78 ± 0.07a 2.48 ± 0.14a,b 1.08 ± 0.04b
a
NF 30 30.08 ± 0.65d 3.93 ± 0.39c
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NF 24 24.41 ± 0.14c 3.16 ± 0.21b,c
4.17 ± 0.31d 18.51 ± 2.07d 230.09 ± 5.58d 1.09 ± 0.13b 3.62 ± 0.13c 1.86 ± 0.08d
Abbreviations are: NF, nanofiltration whey concentrate at 12%, 18%, 24% and 30% dry matter content; DM, dry matter; Na+, sodium; K+, potassium; Ca2+, calcium. Measured values with the same superscript in one row are not statistically different (P ≥ 0.05).
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557 558 559
NF 12 NF 18 a 12.14 ± 0.23 18.32 ± 0.58b 1.45 ± 0.20a 2.41 ± 0.19a,b
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Item DM, % Total protein, % thereof: α-La, g L-1 β-Lg, g L-1 Lactose, g L-1 Na+, g L-1 K+, g L-1 Ca2+, g L-1
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ACCEPTED MANUSCRIPT Table 3.
562
Concentration of remaining native β-Lg and α-La in whey, RO and NF whey concentrates
563
(12-30% DM) after heating up to 125 °C. Data represent mean values ± standard deviations of
564
at least three replicates. a
565 566
Concentration of native α-La (g L-1) 0.60 ± 0.03 1.09 ± 0.01 0.96 ± 0.09 1.15 ± 0.30 1.30 ± 0.15 1.30 ± 0.18 1.69 ± 0.05 1.44 ± 0.38 1.92 ± 0.06
a
SC
Whey RO 12 NF 12 RO 18 NF 18 RO 24 NF 24 RO 30 NF 30
Concentration of native β-Lg (g L-1) 0.82 ± 0.02 1.14 ± 0.20 1.17 ± 0.11 1.12 ± 0.40 1.32 ± 0.25 0.79 ± 0.05 1.50 ± 0.06 0.77 ± 0.26 0.89 ± 0.04
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561
Abbreviations are: RO, reverse osmosis whey concentrate at 12%, 18%, 24% and 30% dry matter content; NF, nanofiltration whey concentrate at 12%, 18%, 24% and 30% dry matter content.
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Table 4.
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Temperature dependent reaction rate constants kT/1.5 for the denaturation of β-Lg in whey, RO
570
and NF whey concentrates. Data represent mean values of three replicates with a 95% confi-
571
dence interval. a
a
Abbreviations are: RO, reverse osmosis whey concentrate at 12%, 18%, 24% and 30% dry matter content; NF, nanofiltration whey concentrate at 12%, 18%, 24% and 30% dry matter content. Measured values with the same superscript in one row are not statistically different (P ≥ 0.05).
M AN U
572 573 574
RI PT
Whey RO 12 NF 12 RO 18 NF 18 RO 24 NF 24 RO 30 NF 30
Temperature dependent reaction rate constants kT/1.5 (10-3 s-1) in whey, RO and NF whey concentrates 90 °C 125 °C 11.7 ± 0.6 a, d 68.2 ± 1.8 a a, d 12.6 ± 2.1 98.9 ± 9.7 b 18.0 ± 0.8 b 45.7 ± 2.7 e a 13.2 ± 1.1 85.6 ± 6.4 b, c 18.3 ± 0.4 b 65.9 ± 5.5 a, d b 19.9 ± 1.2 79.0 ± 9.8 a, c 30.4 ± 1.0 c 65.5 ± 3.5 a, d a 12.7 ± 1.7 60.1 ± 5.0 d 11.2 ± 0.7 d 33.7 ± 4.4 f
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Table 5.
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Temperature dependent reaction rate constants kT/1.0 for the denaturation of α-La in whey, RO
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and NF whey concentrates. Data represent mean values of three replicates with a 95% confi-
579
dence interval. a
a
Abbreviations are: RO, reverse osmosis whey concentrate at 12%, 18%, 24% and 30% dry matter content; NF, nanofiltration whey concentrate at 12%, 18%, 24% and 30% dry matter content. Measured values with the same superscript in one row are not statistically different (P ≥ 0.05).
M AN U
580 581 582
RI PT
Whey RO 12 NF 12 RO 18 NF 18 RO 24 NF 24 RO 30 NF 30
Temperature dependent reaction rate constants kT/1.0 (10-3 s-1) in whey, RO and NF whey concentrates 90 °C 125 °C 3.1 ± 0.3 a 13.2 ± 3.0 a a 3.2 ± 0.3 43.1 ± 8.5 b, c, e 4.9 ± 0.4 b, c 20.3 ± 1.6 d b 4.4 ± 0.3 42.1 ± 7.0 b, c, e 5.8 ± 0.5 c 32.0 ± 3.6 e b 4.3 ± 0.7 41.0 ± 3.4 b 8.4 ± 0.8 d 32.6 ± 4.5 e b 4.3 ± 0.5 53.8 ± 7.9 c 4.4 ± 0.5 b 20.7 ± 6.1 a, d
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Fig. 1. Degree of denaturation (DD) of β-Lg (A, C) and α-La (B, D) in whey (●), RO (■) and
586
NF (◆) concentrates after heating-up phase to (A, B) 90 °C or (C, D) 125 °C of the thermal
587
treatment as affected by the DM content. Data points represent means values ± standard devi-
588
ations of three replicates.
589
Fig. 2. Denaturation of β-Lg in whey (○) and RO whey concentrates with 12% (□), 18% (△),
590
24% (▽) and 30% (◇) DM as a reaction of the order of 1.5 at (A) 90 °C and (B) 125 °C. Co-
591
efficients of determination are for 90 °C: ○, R2 = 0.983; □, R2 = 0.953; △, R2 = 0.926;
592
0.981; ◇, R2 = 0.963. Coefficients of determination are for 125 °C: ○, R2 = 0.979; □, R2 =
593
0.922; △, R2 = 0.929;
594
three replicates with standard deviation.
595
Fig. 3. Denaturation of β-Lg in NF whey concentrates with 12% (□), 18% (△), 24% (▽) and
596
30% (◇) DM as a reaction of the order of 1.5 at (A) 90 °C and (B) 125 °C. Coefficients of
597
determination are for 90 °C: □, R2 = 0.977; △, R2 = 0.995; ▽, R2 = 0.989; ◇, R2 = 0.963. Co-
598
efficients of determination are for 125 °C: □, R2 = 0.980; △, R2 = 0.926;
599
R2 = 0.897. Data points represent mean values of at least three replicates with standard devia-
600
tion.
601
Fig. 4. Denaturation of α-La in whey (○) and RO whey concentrates with 12% (□), 18% (△),
602
24% (▽) and 30% (◇) DM as a reaction of the order of n=1.0 at (A) 90 °C and (B) 125 °C.
603
Coefficients of determination are for 90 °C: ○, R2 = 0.962; □, R2 = 0.949; △, R2 = 0.984;
604
R2 = 0.943; ◇, R2 = 0.968. Coefficients of determination are for 125 °C: ○, R2 = 0.912; □,
605
R2 = 0.933; △, R2 = 0.957;
606
of at least three replicates with standard deviation.
607
Fig. 5. Denaturation of α-La in NF whey concentrates with 12% (□), 18% (△), 24% (▽) and
608
30% (◇) DM as a reaction of the order of n=1.0 at (A) 90 °C and (B) 125 °C. Coefficients of
609
determination are for 90 °C: □, R2 = 0.979; △, R2 = 0.980; ▽, R2 = 0.978; ◇, R2 = 0.973. Co-
610
efficients of determination are for 125 °C: □, R2 = 0.990; △, R2 = 0.977;
611
R2 = 0.906. Data points represent mean values of at least three replicates with standard devia-
612
tion.
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584
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0.984; ◇, R2 = 0.922. Data points represent mean values of at least
▽,
R2 = 0.981; ◇,
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▽,
▽,
▽,
▽,
R2 = 0.960; ◇, R2 = 0.960. Data points represent mean values
▽,
R2 = 0.969; ◇,
24
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