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isolate by heating in the presence of cysteine
Journal Pre-proof Modifying whey protein concentrate/isolate by heating in the presence of cysteine Christina Streicher, Nguyen H.A. Nguyen, Skelte G. Anema PII:
S0958-6946(20)30045-5
DOI:
https://doi.org/10.1016/j.idairyj.2020.104675
Reference:
INDA 104675
To appear in:
International Dairy Journal
Received Date: 26 November 2019 Revised Date:
28 January 2020
Accepted Date: 29 January 2020
Please cite this article as: Streicher, C., Nguyen, N.H.A., Anema, S.G., Modifying whey protein concentrate/isolate by heating in the presence of cysteine, International Dairy Journal, https:// doi.org/10.1016/j.idairyj.2020.104675. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Ltd.
Modifying whey protein concentrate/isolate by heating in the presence of cysteine
Christina Streichera, Nguyen H. A. Nguyena, Skelte G. Anemaa,b,*
a
Fonterra Research and Development Centre, Private Bag 11029, Dairy Farm Road,
Palmerston North 4442, New Zealand b
Riddet Institute, Massey University, Private Bag 11222, Palmerston North 4442, New
Zealand
* Corresponding author. Tel.: +64 6 350 4649 E-mail address: [email protected] (S. G. Anema)
___________________________________________________________________________ ABSTRACT
Heating whey protein concentrate (WPC) or isolate (WPI) solutions at 63 °C and in the presence of cysteine (CYS) denatured α-lactalbumin. The denaturation of α-lactalbumin was described as a first order reaction, with the rate constant increasing linearly with the log of the CYS concentration. Acidifying the WPC/WPI solutions that were heated in the presence of CYS produced a precipitate of denatured α-lactalbumin and a serum containing the native whey proteins. These precipitates/serums could be separated (centrifugation or ultrafiltration/microfiltration) and dried to produce WPC and WPI powders that were either enriched in β-lactoglobulin/depleted in α-lactalbumin or enriched in denatured αlactalbumin/depleted in β-lactoglobulin, although the latter was difficult to dry. When added to skim milk before heating and acidification, β-lactoglobulin-enriched WPC markedly increased the stiffness of the acid gels; β-lactoglobulin-enriched WPI slightly increased the stiffness of the gels. The powder enriched in denatured α-lactalbumin decreased the stiffness of the acid gels. ___________________________________________________________________________
1.
Introduction
The whey proteins in milk are globular proteins that denature when heated above certain temperatures. When denatured, the globular structure unfolds exposing reactive groups that are usually buried within the native conformation. Thus, in complex protein mixtures such as those of whey protein solutions and milk, irreversible aggregation can occur if the system is heated at sufficiently high temperatures. Although denaturation is the precursor to aggregation, it is the aggregation reactions that determine the functional properties of the heated protein systems, and these reactions are dependent on many factors such as the concentration/composition of the protein solutions and the temperature, time and rates of heating and subsequent cooling (Anema, 2014; Dannenberg & Kessler, 1988; Law & Leaver, 1997, 1999; Oldfield, Singh, Taylor, & Pearce, 1998). When considering the unfolding of the whey proteins, as measured by techniques such as differential thermal calorimetry, α-lactalbumin is the most heat labile whey protein unfolding at temperatures below 70 °C (Paulsson & Dejmek, 1990; Paulsson, Hegg, & Castberg, 1985; Ruegg, Moor, & Blanc, 1977). However, as α-lactalbumin does not have a free thiol group, when the temperature is subsequently reduced, the α-lactalbumin will refold to its native conformation. This unfolding/refolding even occurs at higher temperatures (> 70 °C) as long as no unfolded β-lactoglobulin or other unfolded thiol-bearing protein is present to interact with the thiol groups of the unfolded α-lactalbumin (Ruegg et al., 1977). In contrast, β-lactoglobulin is one of the most heat stable of the whey proteins, not unfolding until the temperature exceeds about 70 °C (Paulsson & Dejmek, 1990; Paulsson et al., 1985; Ruegg et al., 1977). When β-lactoglobulin unfolds, it exposes its free thiol group and this can interact with the thiol groups and/or disulphide bonds of other unfolded βlactoglobulin molecules or other proteins that have exposed thiol groups or disulphide bonds,
forming aggregated products. As a consequence, the unfolding of β-lactoglobulin is essentially irreversible. and when α-lactalbumin is present and the sample is heated at temperatures above about 70 °C, the unfolded β-lactoglobulin will interact with the disulphide bonds of α-lactalbumin, preventing its refolding on subsequent cooling (Ruegg et al., 1977). Thiol reagents can modify the denaturation of the whey proteins when milk or whey protein solutions are heated. For example, reduced glutathione or dihydrolipoic acid increased the rate of denaturation of α-lactalbumin in pure solution and both α-lactalbumin and β-lactoglobulin in whey protein isolate solution, with a greater effect on α-lactalbumin than β-lactoglobulin (Wijayanti, Bansal, Sharma, & Deeth, 2014). The gel strength of heatinduced gels prepared from α-lactalbumin/β-lactoglobulin solutions increased when glutathione was added before heating, and it was proposed that the thiol reagent increased the extent of thiol-disulphide exchange reactions between the proteins thus increasing intermolecular disulphide bonding (Legowo, Imade, & Hayakawa, 1993). In another study it was shown that the denaturation of holo-α-lactalbumin was reversible when heated at 90 °C, unfolding during the heating and refolding on subsequent cooling; however, when the thiol reagent cysteine was added, the denaturation was irreversible as disulphide aggregated complexes formed (Nielsen, Lund, Davies, Nielsen, & Nielsen, 2018). In our previous study it was shown that when low levels of thiol reagents, in particular cysteine, were added to skim milk or whey protein isolate solutions, the irreversible denaturation of α-lactalbumin was induced at temperatures between about 60 and 70 °C whereas β-lactoglobulin hardly denatured under these conditions. At higher temperatures, the denaturation of both α-lactalbumin and β-lactoglobulin were accelerated when the thiol reagents were added. These effects were assumed to be due to the thiol reagents promoting thiol-disulphide exchange reactions with the unfolded whey proteins, and, as α-lactalbumin
unfolded at temperatures below those of β-lactoglobulin, they accelerated the aggregation of α-lactalbumin at temperatures between 60 and 70 °C and of both α-lactalbumin and βlactoglobulin at higher temperatures (Nguyen, Streicher, & Anema, 2018). This study investigated the effect of the thiol reagent cysteine on the denaturation of α-lactalbumin and β-lactoglobulin in concentrated (10 or 15% total solids) whey protein concentrate and isolate solutions when these solutions were heated at 63 °C. After heating, the denatured whey proteins were separated from the remaining native proteins, and the native protein solutions (and one denatured whey protein solution) were spray-dried to form protein powders. The functionality of these powders was tested by using them as a protein top-up in skim milk sample that was subsequently heated then acidified to form acid gels.
2.
Materials and methods
2.1.
Whey protein concentrate and whey protein isolate solutions
Whey protein concentrate (WPC, SureProtein Essential Whey Protein Concentrate 392) and whey protein (WPI, SureProtein Clear Whey Protein Isolate 895) were obtained from Fonterra Cooperative Group, Auckland, New Zealand. The WPC or WPI was added to water at a concentration of 2%, 10% or 15% (w/w) on a solids basis. A small amount of sodium azide (0.02%) was added to the WPC or WPI solutions as a preservative. The samples were stirred for about 30 min at room temperature and then allowed to hydrate at 4 °C for at least 12 h before use.
2.2.
Cysteine hydrochloride solution
A 1 M stock solution L-cysteine hydrochloride (CYS; Sigma-Aldrich, St. Louis, MO, USA) was freshly prepared before use by adding the CYS powder to purified water. The sample was dissolved and used within 15 min of preparation.
2.3.
Small-scale sample preparation and heating
CYS at concentrations between 0 and 20 mM were added to the WPC and WPI solutions. The solutions initially had pH values of ~6.8; however, when CYS was added, the pH decreased by about 0.05 to 0.06 pH units per mM of added CYS. The pH was readjusted to 6.8 with 1 M NaOH. Subsamples of the WPC/CYS or WPI/CYS solutions (6 mL) were transferred into sealable glass vials and either unheated or heated, with continuous rocking, in a temperature-controlled oil bath pre-set at 63 °C. The holding time at 63 °C was between 10 and 30 min. After heating the samples were rapidly cooled in cold water.
2.4.
Large-scale sample preparation and heating
For the 10% WPC and WPI solutions, larger scale experiments were performed at ~10 L scale. A stock solution of 30 L of the WPC and WPI solutions were prepared, and sodium azide (~0.02%) was added. The samples were stirred and fully hydrated overnight at 4 °C. The WPC and WPI solutions were divided into three 10 L sub-samples. One sub-sample was an unheated control, a second sub-sample was heated (63 °C, 20 min) without added CYS, and the third sub-sample had 10 mM CYS added before heating. The sample with CYS added was readjusted to pH 6.8 using 1 M NaOH before heating. For the 2% WPC sample, a 10 L batch was prepared and sodium azide (~0.02%) was added. The sample was stirred and fully
hydrated overnight at 4 °C. After hydration 5 mM of CYS was added and the pH readjusted to 6.8 before heating. Heating was achieved using a purpose-built tubular heat exchanger in one water bath with the temperature set at 68 °C and holding vessels in a second water bath pre-set to 63 °C. The sample was pumped through the heat exchanger at 450 mL min-1, which heated the sample to 63 ± 1 °C at the outlet. The sample was pumped directly into the holding vessels in the water bath at 63 °C and held for 20 min. After heating and holding, the samples were rapidly cooled on ice. For samples that were centrifuged, samples were placed in 500 mL bottles, balanced and centrifuged (Heraeus Multifuge 1S-R centrifuge, Thermo Scientific) at 4500 × g for 10 min. The serum was collected and used further. The pellet was not able to be solubilised and was discarded. For samples that were ultrafiltered or microfiltered, hollow fibre ultrafiltration (30,000 Da) or microfiltration (0.1 µm) membranes were used with the associated pumping equipment (Amicon, Inc., Beverly, MA, U.S.A.). Permeate and retentate were collected in different beakers. Diafiltration was achieved by adding 5 times 1 volume equivalents of water to samples during ultrafiltration/microfiltration. On the final pass, the retentate volume was reduced to about one fifth of the original volume before spray drying.
2.5.
Spray drying
Selected samples were spray dried to a powder using a lab-scale spray dryer (Yamato, GA-32, Tokyo, Japan). The samples were warmed to about 45 °C and transferred to the spray drier using the associated peristaltic pump and a flow rate of about 1 L h-1. The dried powder was collected in the cyclone and deposited in the sample chamber. An inlet temperature of ~175 °C was used and the outlet temperature was between 70 to 74 °C. Collected powders
were sealed in foil-lined bags until use. Sub-samples of the powders were analysed for moisture, total nitrogen (TN) and non-protein nitrogen (NPN) using standard methods (ISO, 2016).
2.6.
Traditional and microfluidic chip sodium dodecyl sulphate polyacrylamide gel electrophoresis
To determine the degree of denaturation of α-lactalbumin and β-lactoglobulin, the samples (1 mL) were mixed with 0.2 M sodium acetate solution at pH 4.00 (1.3 mL), which adjusted the pH of the samples to ~pH 4.6 and induced precipitation of the denatured proteins. The samples were then centrifuged (Centrifuge 5417R, Eppendorf AG, Hamburg, Germany) at 21,000 × g for 3 min to separate the denatured (pellet) and native (supernatant) proteins. The α-lactalbumin and β-lactoglobulin contents of the original samples and their respective supernatants were determined using traditional or microfluidic chip sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as has been described in detail previously (Anema, 2009).
2.7.
HPLC-analysis
To determine the native whey protein content of the spray dried powder samples in more detail a HPLC technique was used as has been described in detail previously (Elgar et al., 2000) using a reversed phase HPLC column (Resource RPC 1mL, GE Healthcare, Sweden) with UV detection at 214 nm. Samples were accurately diluted in water to give protein concentration of about 2 mg mL-1 and transferred into HPLC vials and stored at 5 °C until the programme was started. The injection volume was 25 µL and a flowrate of 1 mL
min-1 was used. Details of the solvents and gradients have been given in detail previously (Elgar et al., 2000). Briefly, two solvents were used: solvent A (0.1%, v/v, trifluroacetic acid (TFA) in water) and solvent B (0.09%, v/v TFA, 90%, v/v, acetonitrile in Milli-Q water). The column was initially equilibrated in an 80% to 20% mixture of solvent A to B before sample injection. This was followed by a series of linear gradients with percentage of solvent B increasing to 40% between minutes 1 and 6; to 45% between minutes 6 and 16, to 50% between minutes 16 and 19, held at 50% between minutes 19 to 20 min, increasing to 70% between minutes 20 to 23 and finally to 100% between minutes 23 to 24 and holding at 100% for an additional 1 min. The chromatograms were integrated, and the protein concentrations were calculated automatically using appropriate standard curves for each protein.
2.8.
Acid gelation
The rheological properties of the samples during acidification were measured at 30 °C using a US200 rheometer and a cone (5 cm, 2°) and plate geometry (Paar Physica, Graz, Austria). Skim milk of 10% total solids (w/w) was prepared by mixing low heat skim milk powder (Fonterra Cooperative Group, Auckland, New Zealand) with water. A small amount of sodium azide (0.02%, v/v) was added as a preservative and the samples were stirred for 1 h. The spray dried whey protein powders were added directly to the skim milk at a 1% (w/w) protein level, stirred and allowed to equilibrate for 12 h. Sub samples of the original skim milk or protein fortified skim milk were heated at 80 °C for 30 min with continuous rocking in a temperature-controlled oil bath. After heating the samples were rapidly cooled down in cold water. Glucono-δ-lactone (GDL) was added to the milk at 2% (w/w), and the sample mixed before transferring to the rheometer plate and lowering the cone into position. To prevent evaporation during the measurement, oil was applied on the edge of the sample. The
sample was oscillated at a frequency of 0.1 Hz and a strain of 0.5. Constant measurements of the storage modulus (G’) were taken during 180 min of acidification. A separate sample with GDL added was placed in a water bath at 30 °C and the pH change with time was monitored using a standard combination glass pH electrode.
3.
Results
Two whey protein products were used in the study, a WPC and a WPI. The WPC was a whey protein concentrate produced from cheese whey through ultrafiltration before spray drying. The WPC contains β-lactoglobulin as the major protein component along with high levels of α-lactalbumin and glyco (or caseino) macropeptide (GMP) and a range of minor protein components. In contrast, the WPI is produced from cheese whey by a combination of ion exchange and ultrafiltration processes before spray drying. WPI contains very high levels of β-lactoglobulin, along with a significant level of α-lactalbumin and only very low levels of the other whey proteins. The protein compositional differences between the WPC and WPI can be seen in Fig. 4. The lower level of β-lactoglobulin, the presence of the GMP, and other compositional differences in the WPC when compared with the WPI results in markedly different functional properties, especially heat induced gelation of the whey protein systems at neutral or acidic conditions (Lorenzen & Schrader, 2006; Svanborg, Johansen, Abrahamsen, Schüller, & Skeie, 2016; Veith & Reynolds, 2004; Xianghe, Pan, Peilong, Ismail, & Voorts, 2012). Therefore, it was of interest to compare the impact of CYS on the denaturation, separation and properties of the WPC and WPI samples. There was no detectable denaturation of whey proteins in the unheated samples regardless of the level of CYS added. Denaturation was only observed in samples that were heated, and the level of denaturation was dependent on the particular protein of interest (α-
lactalbumin or β-lactoglobulin), the level of added CYS and the duration of heating at 63 °C (Fig. 1). This is consistent with our previous investigation (Nguyen et al., 2018).
3.1.
Small scale experiments
Preliminary experiments were conducted to determine the conditions to use in subsequent larger scale experiments. In the first experiments, the whey protein solutions (10 and 15%, w/w) were heat-treated at 63 °C for 10 to 30 min with 0 to 20 mM of added CYS. For the WPC, all the β-lactoglobulin remained native, regardless of the CYS level, the holding time at 63 °C or the WPC concentration (Fig. 1A,B). In contrast, the level of denatured α-lactalbumin was dependent on the CYS level, the holding time at 63 °C and the WPC concentration. No denaturation of α-lactalbumin was observed in the heated sample with no CYS added. However, in samples with CYS added, there were higher levels of denaturation at the higher CYS concentrations, longer holding times, and at the higher WPC concentrations. For the 10% WPC solutions with 10 mM CYS, about 55%, 75% and 85% of the α-lactalbumin was denatured after heating for 10, 20 or 30 min respectively, with the levels increasing to 80, 90 and 95% for the 15% WPC solutions after 10, 20 or 30 min respectively (Fig. 1A,B). As with the WPC, no denaturation of β-lactoglobulin was observed for the WPI samples regardless of the WPI concentration, the holding time at 63 °C or the level of added CYS. Similarly, no denaturation of α-lactalbumin was observed in the heated samples without CYS added. However, the denaturation of α-lactalbumin was more extensive than for the WPC samples when CYS was added, and a similar behaviour was observed at both WPI concentrations. At 2 mM added CYS and 10 min heating, about 60% of the α-lactalbumin was denatured, and this increased to more than 80% at longer heating times or at the higher CYS
concentrations. For the WPI samples heated for 10 and 20 min, very low levels (≤ 2%) of native α-lactalbumin could be detected at CYS levels ≥ 7 mM, whereas for samples heated for 30 min, very low levels (≤ 2%) of native α-lactalbumin could be detected at CYS levels ≥ 5 mM (Fig. 1C,D). As the denaturation of α-lactalbumin in WPC at each CYS concentration was conducted at three holding times (600, 1200 and 1800 s), using equation 1 (with the assumption that the denaturation of α-lactalbumin followed first order reaction kinetics), the natural logarithm of the denaturation level of α-lactalbumin in the WPC solutions (Ct/C0, where C0 is the initial α-lactalbumin concentration and Ct is the α-lactalbumin concentration after heating for time (t)) was plotted against the holding time (t) in seconds (Fig. 2A,B). According to equation 1, the rate constants (k1) were obtained from the slopes of the straight lines (-k1). Interestingly, a linear relationship was observed when the rate constants were plotted against the log of the CYS concentration (Fig. 2E). The denaturation of α-lactalbumin in the WPI samples was more rapid than in the WPC samples. As some samples had very low levels (≤ 2%) of native α-lactalbumin remaining, these could not be reliably used in the kinetic evaluation. Consequently, only a limited number of the results were used for determining the rate constants, which reduces the reliability of this kinetic evaluation (Fig. 2C,D). Despite this, the obtained rate constants showed a linear increase with the log of the CYS concentrations, as was observed for the WPC samples (Fig. 2E). These results confirmed that, at each CYS concentration, the slowest rate of α-lactalbumin denaturation was for the 10% WPC sample and the highest rate of α-lactalbumin denaturation was for the 10% and 15% WPI samples (both similar), with the 15% WPC sample intermediate between these. = − . (1)
3.2.
Large scale experiments
From the results of the small-scale experiments, 10% WPC and WPI solutions with CYS concentrations of 0 and 10 mM and a heating temperature of 63 °C and a holding time of 20 min were used for the larger (10 L) scale experiments. After heating and cooling, the original unheated and the heated WPC and WPI samples were adjusted to pH 4.6 and centrifuged and the supernatants collected. The level of native α-lactalbumin and βlactoglobulin in the samples determined by microfluidic chip SDS PAGE. No α-lactalbumin or β-lactoglobulin was denatured in the samples heated in the absence of CYS, whereas in the sample with 10 mM CYS, virtually all the α-lactalbumin but very little β-lactoglobulin was denatured (Fig. 3), consistent with the results of the small-scale experiments. The supernatants were readjusted to pH 6.8 and spray dried to produce dried powders. A total of six powders were produced: WPC and WPI powders from the supernatants of the unheated samples (denoted WPCcontrol-10 and WPIcontrol-10 respectively), WPC and WPI powders from the supernatants of the heated samples (denoted WPCheated-10 and WPIheated-10 respectively), and WPC and WPI powders from the supernatants of heated samples with CYS added and thus enriched in β-lactoglobulin (denoted WPCβ-LG-10 and WPIβ-LG-10 respectively). The moisture, NPN, TN and calculated protein concentrations of the powders are shown in Table 1. The moisture ranged from 7.3 to 8.4%, which is higher than typical commercial powders, probably due to the milder dying conditions used. The total nitrogen levels of the WPC powders, both “as is” or on a dry basis were similar, as were those of the WPI powders; although the TN was slightly lower for the WPCβ-LG-10 and WPIβ-LG-10. As αlactalbumin has higher nitrogen levels than β-lactoglobulin (Grappin & Ribadeau-Dumas, 1992) its removal will lower the overall nitrogen content of the dried powder. In addition, due
to the removal of α-lactalbumin, the proportion of protein to non-protein components will decrease resulting in a lower nitrogen content of the dry powders. In these experiments the residual CYS was not removed from the supernatants prior to drying and therefore the nonprotein nitrogen contents were higher for WPCβ-LG-10 and WPIβ-LG-10 than for the other WPC and WPI samples. Attempts were made to re-disperse the α-lactalbumin-enriched pelleted material from the heated samples with CYS added; however, the pellet was firm and could not be fully re-dispersed and could not be spray dried. The WPC and WPI powders were reconstituted in water and analysed for protein content by HPLC, which gave a more detailed breakdown of the protein composition of each powder. The protein composition of the WPCcontrol-10 and WPCheated-10 samples were similar indicating that the heating had little effect on all proteins present, including the more heat labile IgG and BSA (Fig. 4). For the WPCβ-LG-10 sample, virtually no α-lactalbumin was present, and the BSA level was also markedly reduced compared with the unheated and heated control samples. Overall about 85% of the calculated protein content ((TNNPN)*6.38) was accounted for by this protein analysis, indicating some other protein species may be present in the WPC. For the WPIcontrol-10 and WPIheated-10 samples, β-lactoglobulin was the dominant protein with lower levels of α-lactalbumin, and only very low levels of the other whey proteins. For the WPIβ-LG-10 sample, high levels of β-lactoglobulin were present and only very low levels of α-lactalbumin and the other whey proteins were detected. About 95% of the calculated protein content ((TN-NPN)*6.38) was accounted for by this protein analysis, indicating only low levels of other protein species were present in the WPI. Skim milk samples and skim milk samples with 1% protein (based on (TNNPN)*6.38) from the WPC and WPI powders were heated at 80 °C for 30 min, cooled and then slowly acidified with GDL to form gels. The pH changes and the rheological properties of the samples were measured during acidification. The pH decline after addition of GDL
was very similar for all samples including the skim milk (Fig. 5), which indicates that the added WPC and WPI did not significantly modify the buffering of the milk. This is consistent with previous reports on acid gelation of skim milk samples fortified with various whey protein samples (Anema, 2018, 2019; Nguyen, Wong, Havea, Guyomarc’h, & Anema, 2013). Therefore, any differences in acid gelation are not due to pH differences during acidification. Skim milk produced an acid gel with a final stiffness (after 3 h of gelation) of about 190 Pa. Addition of the WPCcontrol-10 and WPCheated-10 powders to the milk samples increased the G’ to about 250Pa (Fig. 5A). When the WPCβ-LG-10 powder was added to milk before gelation, the final stiffness was substantially higher at about of 400 Pa (Fig. 5A), indicating a significant improvement in acid gelation properties compared with the standard WPCcontrol-10. The addition of 1% protein from WPI powders to skim milk prior to heat treatment and acid gelation caused a marked increase in the final G’, from 190 Pa in the gelled skim milk to about 550 Pa in the gelled skim milk with 1% added WPI. Unlike the WPC samples, there were only small differences in the final G’ between the WPIcontrol-10, WPIheated-10 and WPIβ-LG-10 powders (Fig. 5B). The sample with the WPIβ-LG-10 powder added was slightly higher (590Pa) than the other samples. The final G’ from the acid gels with added WPI were much higher than those from the skim milk with added WPC. A further set of experiments was conducted using 2% WPC. Samples had 5 mM CYS added before heating at 63 °C for 20 min. After heating, the pH was adjusted to 4.6 and the precipitate that formed was allowed to settle overnight. The supernatant was gently decanted from the sediment. Microfluidic chip SDS-PAGE was conducted on the supernatant and sediment, which showed that the supernatant was composed of β-lactoglobulin with no detectable α-lactalbumin, whereas the sediment was composed of both α-lactalbumin and βlactoglobulin (Fig. 6). The sediment sample was diafiltered with 5 equivalent volumes of water using a 0.1 µm microfiltration membrane. The retentate and permeate sample from the
sediment was analysed by microfluidic chip SDS-PAGE after each volume of water, and it was evident that the α-lactalbumin level in the retentate increased and the β-lactoglobulin level in the retentate decreased (Fig. 6) and was found in the permeate (result not shown) indicating that the β-lactoglobulin passed through the membrane whereas the (denatured) αlactalbumin did not pass through the membrane. The β-lactoglobulin enriched supernatant was diafiltered with 5 equivalent volumes of water using a 30 kDa ultrafiltration membrane. After diafiltration, the samples were adjusted to pH 6.8 and spray dried. The powder produced was enriched in β-lactoglobulin and denoted WPCβ-LG-2. It was difficult to spray dry the sediment sample as it regularly blocked the drier nozzle, but eventually enough powder was obtained to do some analysis. The powder from the sediment was enriched in denatured α-lactalbumin and denoted as WPCα-LA-2. The moisture, TN and NPN levels of the powders were determined (Table 1). The moisture levels of the WPCβ-LG-2and WPCα-LA-2 powders were lower than the previously prepared WPC powders. Due to the diafiltration, the excess CYS was removed therefore the NPN was lower, and, as the diafiltration removes lactose and other small molecules, the TN and calculated protein levels were higher than in the previous WPC samples. Skim milk was fortified with 1% protein from the WPCβ-LG-2 and WPCα-LA-2 powders and then slowly acidified with GDL to form gels, with the rheological properties followed during the acidification (Fig. 7). The skim milk with protein from WPCβ-LG-2 powder formed a gel with a final stiffness of about 450 Pa which was markedly higher than the gel obtained from skim milk alone (~190 Pa) and similar to that obtained with the WPCβ-LG-10 powder (Fig. 5A). In contrast, the skim milk sample with protein from the WPCα-LA-2 powder produced an acid gel with a final stiffness of about 120 Pa, which was lower than the gel from the skim milk alone and markedly lower than that from the skim milk with protein from the WPCβ-LG-2 powder.
4.
Discussion
At any given heating time at 63 °C, the level of denaturation of α-lactalbumin in the WPC and WPI samples increased as the CYS level increased (Fig. 1). The denaturation of αlactalbumin in skim milk and whey protein solutions is reported to follow pseudo-first order reaction kinetics (Anema & McKenna, 1996; Dannenberg & Kessler, 1988; Kessler & Beyer, 1991), thus an evaluation using first order kinetics was used to determine the rate constants and these were found to increase with the log of the CYS concentration (Fig. 2). The proposed mechanism for the effect on CYS on the denaturation of α-lactalbumin is that when the α-lactalbumin unfolds to a molten globule state at temperatures above about 61 °C, it exposes its disulphide bonds. In the absence of species with reactive free thiol groups, the α-lactalbumin will refold to its native conformation when cooled; however, in the presence of a reactive free thiol group, the thiol will initiate thiol-disulphide exchange reactions causing the α-lactalbumin to aggregate and thus unable to refold. For the denaturation/aggregation reactions of the whey proteins in milk and whey solutions, at temperatures below ~80 °C for α-lactalbumin and at temperature below ~90 °C for β-lactoglobulin, the unfolding of the protein is considered to be the rate determining step, with the subsequent aggregation reaction occurring rapidly. For α-lactalbumin at 63 °C, the addition of CYS is unlikely to change the rate of unfolding of the protein, but will increase the propensity of aggregation by initiating thiol-disulphide exchange reactions, allowing for irreversible aggregation to occur. Although only low levels of free thiols would be required to initiate the thiol-disulphide exchange reactions, due to the high pKa (~8.5) of the thiol group of free CYS (Netto et al., 2007) the concentration of the reactive thiolate group (S-) in CYS will be very low at the pH used in these experiments thus an increasing rate of aggregation
would be expected with increasing CYS concentrations until sufficient S- is introduced relative to the number of disulphide bonds (Figs. 1 and 2). Although α-lactalbumin is the predominant protein denatured in WPC and WPI when heated in the presence of CYS (Figs. 3 and 4), the minor whey protein BSA was also denatured under these heating conditions (Fig. 4). The denaturation temperature of BSA is lower than that of α-lactalbumin at about 62 °C (Paulsson et al., 1985; Ruegg et al., 1977), indicating that it is unfolding at the heating conditions used. However, the observation that it is not irreversibly denatured on heating indicates that the free thiol in BSA is not reactive at these low temperatures, although it probably is exposed and reactive at higher temperatures as BSA denaturation is irreversible when cycled between 20 and 110 °C (Ruegg et al., 1977). By precipitation and centrifugation (Figs. 3 and 4) or ultrafiltration/microfiltration with diafiltration (Fig. 6) and subsequent spray drying, it was possible to produce modified whey protein powders. Addition of the WPCcontrol-10 and WPCheated-10 powders to skim milk before heating and acidification increased the stiffness of the acid gels by about 30% (Fig. 5A). A similar, although larger increase in stiffness in acid gels was observed when a different type of WPC was added to skim milk at similar addition levels prior to heating and acidification (Lucey, Munro, & Singh, 1999). The results of this previous study are not directly comparable as the GDL level was lower and the WPC used was optimised for gelation and cultured applications (Fenwick & MacGibbon, 2014); however, these results demonstrate that the stiffness of acid gels is increased when whey protein concentrates are added to milk. When the WPIcontrol-10 and WPIheated-10 powders were added to the milk before heating and acidification the stiffness of the gels increased by about 250% (Fig. 5B), substantially more than when the WPC was added (Fig. 5A). Unlike WPC, which contains β-lactoglobulin, α-lactalbumin and GMP at significant levels, the WPI is composed predominantly of β-
lactoglobulin with a lower proportion of α-lactalbumin, and virtually no GMP (Fig. 4). As βlactoglobulin contributes more to the acid gel stiffness than α-lactalbumin (Graveland-Bikker & Anema, 2003), the much higher proportion of β-lactoglobulin in the WPI than the WPC would probably account for the larger increase in stiffness of the acid gels prepared from milk fortified with WPI. When the WPCβ-LG-10 powder was added to the milk before heating and acidification, a marked increase in acid gel stiffness was observed (Fig. 5A). This WPCβ-LG-10 powder contained residual CYS and previous studies have shown that the stiffness of acid gels prepared from skim milk could be markedly increased by the addition of thiol reagents (Nguyen et al., 2013). However, the level of residual CYS in the milk would be low (less than 1 mM) and unlikely to have a significant effect at these low concentrations as previous studies have shown that thiol reagents at these low concentrations had only small effects on the acid gelation properties of heated milk (Nguyen et al., 2013). In addition, a very similar increase in gel stiffness was obtained when the diafiltered WPCβ-LG-2 powder was used (Fig. 7), suggesting that the increase in stiffness was due to the removal of the α-lactalbumin and thereby increasing the proportion β-lactoglobulin in the sample. The acid gel containing the WPCα-LA-2 powder sample had a lower stiffness consistent with reports that show denatured/aggregated whey proteins have a detrimental effect on acid gel properties (Lucey, Tamehana, Singh, & Munro, 1998; Schorsch, Wilkins, Jones, & Norton, 2001). In contrast to the WPCβ-LG-10 and WPCβ-LG-2 powders, when the WPIβ-LG-10 powder was added to milk before heating and acidification, only a small increase in acid gel stiffness was observed (Figure 5B). The WPIcontrol-10 and WPIheated-10 powders have a high level of βlactoglobulin and a proportionally low level of α-lactalbumin (Fig. 4), and WPI addition to milk markedly increases acid gel stiffness compared with WPC (Fig. 5). Therefore, the
removal of the α-lactalbumin from the WPI appears to have only a small effect on the WPI performance in an acid gel system.
5.
Conclusion
Addition of CYS to concentrated (10 and 15%, w/w) WPC and WPI solutions before heating at 63 °C resulted in the denaturation of α-lactalbumin with β-lactoglobulin remaining native. The level of denaturation of α-lactalbumin increased with the level of added CYS at each heating time and with heating time at each level of added CYS. As α-lactalbumin unfolds at temperatures above 60 °C whereas β-lactoglobulin unfolds at temperatures above 70 °C, the CYS can initiate thiol-disulphide exchange reactions with the disulphide bonds of the unfolded α-lactalbumin at 63 °C producing intermolecular disulphide bonded aggregates, thus preferentially denaturing the α-lactalbumin. The denaturation of α-lactalbumin in the presence of CYS could be described as a (pseudo) first order reaction and the denaturation rate constant increased linearly with the log of the CYS concentration, indicating that the CYS accelerated the irreversible denaturation and aggregation of the α-lactalbumin. WPC solutions at 2% and 10% (w/w) and WPI solutions of 10% (w/w) that were heated in the presence of CYS could be separated into precipitates containing denatured αlactalbumin and solutions containing the remaining native whey proteins, in particular βlactoglobulin. These could be spray dried producing modified WPC and WPI powders that were either depleted in α-lactalbumin/enriched in β-lactoglobulin (WPCβ-LG-10, WPCβ-LG-2, and WPIβ-LG-10) or enriched in denatured α-lactalbumin (WPCα-LA-2). Although some difficulty was encountered drying the WPCα-LA-2 due to nozzle blockages, these results indicate that WPC and WPI products with modified protein compositions could be prepared by preferential denaturation and separation of α-lactalbumin from the other whey proteins.
Surprisingly, when the WPIcontrol-10, WPIheated-10, and WPIβ-LG-10 were added to skim milk, heated and acidified, the gels formed had similar properties. This is probably due to the WPIcontrol-10 and WPIheated-10 having high levels of β-lactoglobulin and markedly increasing acid gel stiffness so that minimal further improvement was achieved by increasing the βlactoglobulin level in the WPIβ-LG-10. In contrast, markedly different gel properties were obtained when the WPCcontrol-10, the WPIheated-10, the WPCβ-LG-10, the WPCβ-LG-2, or the WPCαLA-2
were added to the skim milk before heating and acidification, with the WPCα-LA-2
decreasing the acid gel stiffness and the WPCβ-LG-10 and WPCβ-LG-2 increasing the acid gel stiffness compared with the WPCcontrol-10 and WPCheated-10, which were similar. The WPCcontrol-10 and WPCheated-10 have lower levels of β-lactoglobulin than WPIcontrol-10 and WPIheated-10, and only causes moderate increases in acid gel stiffness, thus increasing the βlactoglobulin level in the WPCβ-LG-10 and WPCβ-LG-2 powders may have a more marked effect on the acid gels than for the WPI samples. As the α-lactalbumin in the WPCα-LA-2 is already denatured and aggregated, it probably does not contribute to the acid gel structure and thus a gel with decreased stiffness is obtained. Overall these results indicate that WPC samples with modified functional properties could be prepared by the preferential denaturation and separation of α-lactalbumin through reaction with CYS.
References
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Singh (Eds.), Milk proteins (2nd edn., pp. 269–318). San Diego, CA, USA: Academic Press. Anema, S. G. (2018). Effect of whey protein addition and pH on the acid gelation of heated skim milk. International Dairy Journal, 79, 5–14. Anema, S. G. (2019). Acidification of lactoferrin-casein micelle complexes in skim milk. International Dairy Journal, 99, Article 104550. Anema, S. G., & McKenna, A. B. (1996). Reaction kinetics of thermal denaturation of whey proteins in heated reconstituted whole milk. Journal of Agricultural and Food Chemistry, 44, 422–428. Dannenberg, F., & Kessler, H. G. (1988). Reaction kinetics of the denaturation of whey proteins in milk. Journal of Food Science, 53, 258–263. Elgar, D. F., Norris, C. S., Ayers, J. S., Pritchard, M., Otter, D. E., & Palmano, K. P. (2000). Simultaneous separation and quantitation of the major bovine whey proteins including proteose peptone and caseinomacropeptide by reversed-phase high-performance liquid chromatography on polystyrene–divinylbenzene. Journal of Chromatography A, 878, 183–196. Fenwick, R., & MacGibbon, J. (2014). Whey to go: Whey protein concentrate: A New Zealand success story. Wellington, NZ: Ngaio Press. Grappin, R., & Ribadeau-Dumas, B. (1992). Analytical methods for milk proteins. P. F. Fox In (Ed.), Advanced dairy chemistry. Vol. 1. Proteins (pp. 1–62). London, UK: Elsevier Applied Science. Graveland-Bikker, J. F., & Anema, S. G. (2003). Effect of individual whey proteins on the rheological properties of acid gels prepared from heated skim milk. International Dairy Journal, 13, 401–408.
ISO. (2016). Milk and milk products-Determination of nitrogen content — Part 4: Determination of protein and non-protein nitrogen content and true protein content calculation (Reference method). ISO 8968-4:2016/IDF 20-4:2016. Geneva, Switzerland: International Standards Organization. Kessler, H. G., & Beyer, H. J. (1991). Thermal denaturation of whey proteins and its effect in dairy technology. International Journal of Biological Macromolecules, 13, 165–173. Law, A. J. R., & Leaver, J. (1997). Effect of protein concentration on rates of thermal denaturation of whey proteins in milk. Journal of Agricultural and Food Chemistry, 45, 4255–4261. Law, A. J. R., & Leaver, J. (1999). Factors affecting the heat denaturation of whey proteins in cows’ milk. International Dairy Journal, 9, 407–408. Legowo, A. M., Imade, T., & Hayakawa, S. (1993). Heat-induced gelation of the mixtures of α-lactalbumin and β-lactoglobulin in the presence of glutathione. Food Research International, 26, 103–108. Lorenzen, P. C., & Schrader, K. (2006). A comparative study of the gelation properties of whey protein concentrate and whey protein isolate. Lait, 86, 259–271. Lucey, J. A., Munro, P. A., & Singh, H. (1999). Effects of heat treatment and whey protein addition on the rheological properties and structure of acid skim milk gels. International Dairy Journal, 9, 275–279. Lucey, J. A., Tamehana, M., Singh, H., & Munro, P. A. (1998). Effect of interactions between denatured whey proteins and casein micelles on the formation and rheological properties of acid skim milk gels. Journal of Dairy Research, 65, 555– 567. Netto, L. E. S., de Oliveira, M. A., Monteiro, G., Demasi, A. P. D., Cussiol, J. R. R., Discola, K. F., et al. (2007). Reactive cysteine in proteins: Protein folding, antioxidant defense,
redox signaling and more. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 146, 180–193. Nguyen, N. H. A., Streicher, C., & Anema, S. G. (2018). The effect of thiol reagents on the denaturation of the whey protein in milk and whey protein concentrate solutions. International Dairy Journal, 85, 285–293. Nguyen, N. H. A., Wong, M., Havea, P., Guyomarc’h, F., & Anema, S. G. (2013). The protein interactions and rheological properties of skim milk heated in the presence of low levels of reducing agent. Food Chemistry, 138, 1604–1609. Nielsen, L. R., Lund, M. N., Davies, M. J., Nielsen, J. H., & Nielsen, S. B. (2018). Effect of free cysteine on the denaturation and aggregation of holo α-lactalbumin. International Dairy Journal, 79, 52–61. Oldfield, D. J., Singh, H., Taylor, M., & Pearce, K. (1998). Kinetics of denaturation and aggregation of whey proteins in skim milk heated in an ultra-high temperature (UHT) pilot plant. International Dairy Journal, 8, 311–318. Paulsson, M., & Dejmek, P. (1990). Thermal-denaturation of whey proteins in mixtures with caseins studied by differential scanning calorimetry. Journal of Dairy Science, 73, 590–600. Paulsson, M., Hegg, P. O., & Castberg, H. B. (1985). Thermal stability of whey proteins studied by differential scanning calorimetry. Thermochimica Acta, 95, 435–440. Ruegg, M., Moor, U., & Blanc, B. (1977). A calorimetric study of thermal denaturation of whey proteins in simulated milk ultrafiltrate. Journal of Dairy Research, 44, 509–520. Schorsch, C., Wilkins, D. K., Jones, M. G., & Norton, I. T. (2001). Gelation of casein-whey mixtures: effects of heating whey proteins alone or in the presence of casein micelles. Journal of Dairy Research, 68, 471–481.
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Figure legends
Fig. 1. Effect of CYS concentration on the level of native β-lactoglobulin ( , ▲, lactalbumin ( , ▲,
) and α-
) in 10% WPC (A), 15% WPC (B), 10% WPI (C) or 15% WPI (D)
solutions heated at 63 °C for 10 ( ,
), 20 (▲, ▲) or 30 ( ,
) min. Native protein levels
were determined by traditional SDS-PAGE.
Fig. 2. Denaturation of α-lactalbumin WPC (A, B) and WPI (C, D) solutions as a first order reaction. A, 10% WPC; B, 15% WPC; C, 10% WPI; D, 15% WPI with 2 mM ( ), 5 mM ( ), 7 mM (▲), 10 mM (▲) or 20 mM ( ) added CYS. Samples were heated at 63 °C for different times. For the WPC samples (A, B) all data were used for obtaining rate constants; for the WPI samples (C, D) only samples that the lines pass through were used for obtaining rate constants (see text for explanation). E: rate constants plotted against CYS concentration for 10% WPC ( ), 15% WPC ( ), 10% WPI (▲) and 15% WPI (▲).
Fig. 3. Level of native β-lactoglobulin (red bars) and α-lactalbumin (blue bars) in unheated and heated (63 °C, 20 min) 10% WPC and 10% WPI samples. Native protein levels were determined by microfluidic chip SDS PAGE.
Fig. 4. Protein composition of spray dried WPC and WPI powders from unheated and heated (63 °C, 20 min) samples as determined by reversed-phase HPLC. Red bars: β-lactoglobulin, blue bars: α-lactalbumin, green bars: glycomacropeptide, black bars: BSA, pink bars: PP5, teal bars: IgG.
Fig. 5. Gelation curves showing the change in stiffness and pH with time after 2% GDL was added to heated (80 °C, 30 min) milk samples. A: Acid gels were prepared from skim milk ( , ▲), skim milk with 1% protein from WPCcontrol-10 ( , ▼), WPCheated-10 (▼) or WPCβ-LG10
(▲). B: Acid gels were prepared from skim milk ( , ▲), skim milk with 1% protein from
WPIcontrol-10 ( , ▼), WPIheated-10 (▼) or WPIβ-LG-10 (▲).
Fig. 6. Protein composition of the supernatants, sediments, and sediment retentates during diafiltration with 1 (R1), 2 (R2), 3 (R3), 4 (R4), and 5 (R5) equivalent volumes of water. Supernatants and sediments were obtained from 2% WPC heated in the presence of 5 mM of CYS. Red bars: β-lactoglobulin, blue bars: α-lactalbumin. Native protein levels were determined by microfluidic chip SDS-PAGE.
Fig. 7. Gelation curves showing the change in stiffness with time after 2% GDL was added to heated (80 °C, 30 min) skim milk ( ), skim milk with 1% protein from WPCβ-LG-2 ( ), or skim milk with 1% protein from WPCα-LA-2 (▼).
1
Table 1
2
Moisture, total nitrogen, non-protein nitrogen and calculated protein concentrations of WPC and WPI powders. a Powder descriptor
Powder sample starting material
Moisture (%, w/w)
Total nitrogen (%, w/w)
Non-protein nitrogen (%, w/w)
Protein (TN-NPN)*6.38 (%, w/w)
WPCcontrol-10
10% WPC unheated
7.7
11.69 (12.67)
0.657 (0.712)
70.39 (76.26)
WPCheated-10
10% WPC heated
7.3
11.65 (12.57)
0.897 (0.968)
68.60 (74.01)
WPCβ-LG-10
10% WPC/10 mM CYS supernatant heated
8.2
11.16 (12.16)
1.26 (1.373)
63.16 (68.80)
WPIcontrol-10
10% WPI unheated
8.3
13.81 (15.06)
0.098 (0.107)
87.48 (95.40)
WPIheated-10
10% WPI heated
7.4
13.93 (15.04)
0.086 (0.093)
88.32 (95.38)
WPIβ-LG-10
10% WPI/10 mM CYS supernatant heated
8.4
13.57 (14.81)
0.330 (0.360)
84.47 (92.22)
WPCβ-LG-2
2% WPC/5 mM CYS heated supernatant
4.8
13.61 (14.30)
0.356 (0.374)
84.56 (88.82)
WPCα-LA-2
2% WPC/5 mM CYS heated retentate
2.8
12.23 (12.58)
0.213 (0.219)
76.67 (78.88)
3 4
a
Total nitrogen, non-protein nitrogen and calculated protein concentrations presented on an as is basis with values on a dry basis in parentheses.
1
100 80 60
Native protein (%)
40 20 0 100
A
B
C
D
80 60 40 20 0
0
5
10
15
20
0
Cysteine added (mM)
Figure 1
5
10
15
20
0
A
B
C
D
-1 -2
ln(Ct/C0)
-3 -4 0 -1 -2 -3 -4 -5 -6 0
500
1000
1500
0
500
1000
Rate constant (k1 * 1000)
Time (seconds)
E
6
4
2
0.2
0.4
0.6
0.8
1.0
log ([Cysteine])
Figure 2
1.2
1.4
1500
Figure 3
Intensity (arbitrary units) 600
Heated WPI with CYS
500
Heated WPI
400
Unheated WPI
300
Heated WPC with CYS
200
Heated WPC
100
0
Unheated WPC
Figure 4
-1
mg protein. g powder
Heated WPI Heated WPI with CYS
800
Unheated WPI
600
Heated WPC with CYS
400
Heated WPC
200
0
Unheated WPC
A 400
6.5
300
6.0 5.5 5.0
100 4.5 0 600
pH
Stiffness (G' in Pa)
200
B 6.5
500
6.0 400
5.5
300 200
5.0
100
4.5
0
0
50
100
Time (min)
Figure 5
150
Figure 6
Protein composition (%)
R4 R5
100
R3
80
R2
60
R1
40
Sediment
20
0
supernatant
Stiffness (G' in Pa)
400 300 200 100 0 0
50
100
Time (min)
Figure 7
150
S0958-6946(20)30045-5
DOI:
https://doi.org/10.1016/j.idairyj.2020.104675
Reference:
INDA 104675
To appear in:
International Dairy Journal
Received Date: 26 November 2019 Revised Date:
28 January 2020
Accepted Date: 29 January 2020
Please cite this article as: Streicher, C., Nguyen, N.H.A., Anema, S.G., Modifying whey protein concentrate/isolate by heating in the presence of cysteine, International Dairy Journal, https:// doi.org/10.1016/j.idairyj.2020.104675. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Ltd.
Modifying whey protein concentrate/isolate by heating in the presence of cysteine
Christina Streichera, Nguyen H. A. Nguyena, Skelte G. Anemaa,b,*
a
Fonterra Research and Development Centre, Private Bag 11029, Dairy Farm Road,
Palmerston North 4442, New Zealand b
Riddet Institute, Massey University, Private Bag 11222, Palmerston North 4442, New
Zealand
* Corresponding author. Tel.: +64 6 350 4649 E-mail address: [email protected] (S. G. Anema)
___________________________________________________________________________ ABSTRACT
Heating whey protein concentrate (WPC) or isolate (WPI) solutions at 63 °C and in the presence of cysteine (CYS) denatured α-lactalbumin. The denaturation of α-lactalbumin was described as a first order reaction, with the rate constant increasing linearly with the log of the CYS concentration. Acidifying the WPC/WPI solutions that were heated in the presence of CYS produced a precipitate of denatured α-lactalbumin and a serum containing the native whey proteins. These precipitates/serums could be separated (centrifugation or ultrafiltration/microfiltration) and dried to produce WPC and WPI powders that were either enriched in β-lactoglobulin/depleted in α-lactalbumin or enriched in denatured αlactalbumin/depleted in β-lactoglobulin, although the latter was difficult to dry. When added to skim milk before heating and acidification, β-lactoglobulin-enriched WPC markedly increased the stiffness of the acid gels; β-lactoglobulin-enriched WPI slightly increased the stiffness of the gels. The powder enriched in denatured α-lactalbumin decreased the stiffness of the acid gels. ___________________________________________________________________________
1.
Introduction
The whey proteins in milk are globular proteins that denature when heated above certain temperatures. When denatured, the globular structure unfolds exposing reactive groups that are usually buried within the native conformation. Thus, in complex protein mixtures such as those of whey protein solutions and milk, irreversible aggregation can occur if the system is heated at sufficiently high temperatures. Although denaturation is the precursor to aggregation, it is the aggregation reactions that determine the functional properties of the heated protein systems, and these reactions are dependent on many factors such as the concentration/composition of the protein solutions and the temperature, time and rates of heating and subsequent cooling (Anema, 2014; Dannenberg & Kessler, 1988; Law & Leaver, 1997, 1999; Oldfield, Singh, Taylor, & Pearce, 1998). When considering the unfolding of the whey proteins, as measured by techniques such as differential thermal calorimetry, α-lactalbumin is the most heat labile whey protein unfolding at temperatures below 70 °C (Paulsson & Dejmek, 1990; Paulsson, Hegg, & Castberg, 1985; Ruegg, Moor, & Blanc, 1977). However, as α-lactalbumin does not have a free thiol group, when the temperature is subsequently reduced, the α-lactalbumin will refold to its native conformation. This unfolding/refolding even occurs at higher temperatures (> 70 °C) as long as no unfolded β-lactoglobulin or other unfolded thiol-bearing protein is present to interact with the thiol groups of the unfolded α-lactalbumin (Ruegg et al., 1977). In contrast, β-lactoglobulin is one of the most heat stable of the whey proteins, not unfolding until the temperature exceeds about 70 °C (Paulsson & Dejmek, 1990; Paulsson et al., 1985; Ruegg et al., 1977). When β-lactoglobulin unfolds, it exposes its free thiol group and this can interact with the thiol groups and/or disulphide bonds of other unfolded βlactoglobulin molecules or other proteins that have exposed thiol groups or disulphide bonds,
forming aggregated products. As a consequence, the unfolding of β-lactoglobulin is essentially irreversible. and when α-lactalbumin is present and the sample is heated at temperatures above about 70 °C, the unfolded β-lactoglobulin will interact with the disulphide bonds of α-lactalbumin, preventing its refolding on subsequent cooling (Ruegg et al., 1977). Thiol reagents can modify the denaturation of the whey proteins when milk or whey protein solutions are heated. For example, reduced glutathione or dihydrolipoic acid increased the rate of denaturation of α-lactalbumin in pure solution and both α-lactalbumin and β-lactoglobulin in whey protein isolate solution, with a greater effect on α-lactalbumin than β-lactoglobulin (Wijayanti, Bansal, Sharma, & Deeth, 2014). The gel strength of heatinduced gels prepared from α-lactalbumin/β-lactoglobulin solutions increased when glutathione was added before heating, and it was proposed that the thiol reagent increased the extent of thiol-disulphide exchange reactions between the proteins thus increasing intermolecular disulphide bonding (Legowo, Imade, & Hayakawa, 1993). In another study it was shown that the denaturation of holo-α-lactalbumin was reversible when heated at 90 °C, unfolding during the heating and refolding on subsequent cooling; however, when the thiol reagent cysteine was added, the denaturation was irreversible as disulphide aggregated complexes formed (Nielsen, Lund, Davies, Nielsen, & Nielsen, 2018). In our previous study it was shown that when low levels of thiol reagents, in particular cysteine, were added to skim milk or whey protein isolate solutions, the irreversible denaturation of α-lactalbumin was induced at temperatures between about 60 and 70 °C whereas β-lactoglobulin hardly denatured under these conditions. At higher temperatures, the denaturation of both α-lactalbumin and β-lactoglobulin were accelerated when the thiol reagents were added. These effects were assumed to be due to the thiol reagents promoting thiol-disulphide exchange reactions with the unfolded whey proteins, and, as α-lactalbumin
unfolded at temperatures below those of β-lactoglobulin, they accelerated the aggregation of α-lactalbumin at temperatures between 60 and 70 °C and of both α-lactalbumin and βlactoglobulin at higher temperatures (Nguyen, Streicher, & Anema, 2018). This study investigated the effect of the thiol reagent cysteine on the denaturation of α-lactalbumin and β-lactoglobulin in concentrated (10 or 15% total solids) whey protein concentrate and isolate solutions when these solutions were heated at 63 °C. After heating, the denatured whey proteins were separated from the remaining native proteins, and the native protein solutions (and one denatured whey protein solution) were spray-dried to form protein powders. The functionality of these powders was tested by using them as a protein top-up in skim milk sample that was subsequently heated then acidified to form acid gels.
2.
Materials and methods
2.1.
Whey protein concentrate and whey protein isolate solutions
Whey protein concentrate (WPC, SureProtein Essential Whey Protein Concentrate 392) and whey protein (WPI, SureProtein Clear Whey Protein Isolate 895) were obtained from Fonterra Cooperative Group, Auckland, New Zealand. The WPC or WPI was added to water at a concentration of 2%, 10% or 15% (w/w) on a solids basis. A small amount of sodium azide (0.02%) was added to the WPC or WPI solutions as a preservative. The samples were stirred for about 30 min at room temperature and then allowed to hydrate at 4 °C for at least 12 h before use.
2.2.
Cysteine hydrochloride solution
A 1 M stock solution L-cysteine hydrochloride (CYS; Sigma-Aldrich, St. Louis, MO, USA) was freshly prepared before use by adding the CYS powder to purified water. The sample was dissolved and used within 15 min of preparation.
2.3.
Small-scale sample preparation and heating
CYS at concentrations between 0 and 20 mM were added to the WPC and WPI solutions. The solutions initially had pH values of ~6.8; however, when CYS was added, the pH decreased by about 0.05 to 0.06 pH units per mM of added CYS. The pH was readjusted to 6.8 with 1 M NaOH. Subsamples of the WPC/CYS or WPI/CYS solutions (6 mL) were transferred into sealable glass vials and either unheated or heated, with continuous rocking, in a temperature-controlled oil bath pre-set at 63 °C. The holding time at 63 °C was between 10 and 30 min. After heating the samples were rapidly cooled in cold water.
2.4.
Large-scale sample preparation and heating
For the 10% WPC and WPI solutions, larger scale experiments were performed at ~10 L scale. A stock solution of 30 L of the WPC and WPI solutions were prepared, and sodium azide (~0.02%) was added. The samples were stirred and fully hydrated overnight at 4 °C. The WPC and WPI solutions were divided into three 10 L sub-samples. One sub-sample was an unheated control, a second sub-sample was heated (63 °C, 20 min) without added CYS, and the third sub-sample had 10 mM CYS added before heating. The sample with CYS added was readjusted to pH 6.8 using 1 M NaOH before heating. For the 2% WPC sample, a 10 L batch was prepared and sodium azide (~0.02%) was added. The sample was stirred and fully
hydrated overnight at 4 °C. After hydration 5 mM of CYS was added and the pH readjusted to 6.8 before heating. Heating was achieved using a purpose-built tubular heat exchanger in one water bath with the temperature set at 68 °C and holding vessels in a second water bath pre-set to 63 °C. The sample was pumped through the heat exchanger at 450 mL min-1, which heated the sample to 63 ± 1 °C at the outlet. The sample was pumped directly into the holding vessels in the water bath at 63 °C and held for 20 min. After heating and holding, the samples were rapidly cooled on ice. For samples that were centrifuged, samples were placed in 500 mL bottles, balanced and centrifuged (Heraeus Multifuge 1S-R centrifuge, Thermo Scientific) at 4500 × g for 10 min. The serum was collected and used further. The pellet was not able to be solubilised and was discarded. For samples that were ultrafiltered or microfiltered, hollow fibre ultrafiltration (30,000 Da) or microfiltration (0.1 µm) membranes were used with the associated pumping equipment (Amicon, Inc., Beverly, MA, U.S.A.). Permeate and retentate were collected in different beakers. Diafiltration was achieved by adding 5 times 1 volume equivalents of water to samples during ultrafiltration/microfiltration. On the final pass, the retentate volume was reduced to about one fifth of the original volume before spray drying.
2.5.
Spray drying
Selected samples were spray dried to a powder using a lab-scale spray dryer (Yamato, GA-32, Tokyo, Japan). The samples were warmed to about 45 °C and transferred to the spray drier using the associated peristaltic pump and a flow rate of about 1 L h-1. The dried powder was collected in the cyclone and deposited in the sample chamber. An inlet temperature of ~175 °C was used and the outlet temperature was between 70 to 74 °C. Collected powders
were sealed in foil-lined bags until use. Sub-samples of the powders were analysed for moisture, total nitrogen (TN) and non-protein nitrogen (NPN) using standard methods (ISO, 2016).
2.6.
Traditional and microfluidic chip sodium dodecyl sulphate polyacrylamide gel electrophoresis
To determine the degree of denaturation of α-lactalbumin and β-lactoglobulin, the samples (1 mL) were mixed with 0.2 M sodium acetate solution at pH 4.00 (1.3 mL), which adjusted the pH of the samples to ~pH 4.6 and induced precipitation of the denatured proteins. The samples were then centrifuged (Centrifuge 5417R, Eppendorf AG, Hamburg, Germany) at 21,000 × g for 3 min to separate the denatured (pellet) and native (supernatant) proteins. The α-lactalbumin and β-lactoglobulin contents of the original samples and their respective supernatants were determined using traditional or microfluidic chip sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as has been described in detail previously (Anema, 2009).
2.7.
HPLC-analysis
To determine the native whey protein content of the spray dried powder samples in more detail a HPLC technique was used as has been described in detail previously (Elgar et al., 2000) using a reversed phase HPLC column (Resource RPC 1mL, GE Healthcare, Sweden) with UV detection at 214 nm. Samples were accurately diluted in water to give protein concentration of about 2 mg mL-1 and transferred into HPLC vials and stored at 5 °C until the programme was started. The injection volume was 25 µL and a flowrate of 1 mL
min-1 was used. Details of the solvents and gradients have been given in detail previously (Elgar et al., 2000). Briefly, two solvents were used: solvent A (0.1%, v/v, trifluroacetic acid (TFA) in water) and solvent B (0.09%, v/v TFA, 90%, v/v, acetonitrile in Milli-Q water). The column was initially equilibrated in an 80% to 20% mixture of solvent A to B before sample injection. This was followed by a series of linear gradients with percentage of solvent B increasing to 40% between minutes 1 and 6; to 45% between minutes 6 and 16, to 50% between minutes 16 and 19, held at 50% between minutes 19 to 20 min, increasing to 70% between minutes 20 to 23 and finally to 100% between minutes 23 to 24 and holding at 100% for an additional 1 min. The chromatograms were integrated, and the protein concentrations were calculated automatically using appropriate standard curves for each protein.
2.8.
Acid gelation
The rheological properties of the samples during acidification were measured at 30 °C using a US200 rheometer and a cone (5 cm, 2°) and plate geometry (Paar Physica, Graz, Austria). Skim milk of 10% total solids (w/w) was prepared by mixing low heat skim milk powder (Fonterra Cooperative Group, Auckland, New Zealand) with water. A small amount of sodium azide (0.02%, v/v) was added as a preservative and the samples were stirred for 1 h. The spray dried whey protein powders were added directly to the skim milk at a 1% (w/w) protein level, stirred and allowed to equilibrate for 12 h. Sub samples of the original skim milk or protein fortified skim milk were heated at 80 °C for 30 min with continuous rocking in a temperature-controlled oil bath. After heating the samples were rapidly cooled down in cold water. Glucono-δ-lactone (GDL) was added to the milk at 2% (w/w), and the sample mixed before transferring to the rheometer plate and lowering the cone into position. To prevent evaporation during the measurement, oil was applied on the edge of the sample. The
sample was oscillated at a frequency of 0.1 Hz and a strain of 0.5. Constant measurements of the storage modulus (G’) were taken during 180 min of acidification. A separate sample with GDL added was placed in a water bath at 30 °C and the pH change with time was monitored using a standard combination glass pH electrode.
3.
Results
Two whey protein products were used in the study, a WPC and a WPI. The WPC was a whey protein concentrate produced from cheese whey through ultrafiltration before spray drying. The WPC contains β-lactoglobulin as the major protein component along with high levels of α-lactalbumin and glyco (or caseino) macropeptide (GMP) and a range of minor protein components. In contrast, the WPI is produced from cheese whey by a combination of ion exchange and ultrafiltration processes before spray drying. WPI contains very high levels of β-lactoglobulin, along with a significant level of α-lactalbumin and only very low levels of the other whey proteins. The protein compositional differences between the WPC and WPI can be seen in Fig. 4. The lower level of β-lactoglobulin, the presence of the GMP, and other compositional differences in the WPC when compared with the WPI results in markedly different functional properties, especially heat induced gelation of the whey protein systems at neutral or acidic conditions (Lorenzen & Schrader, 2006; Svanborg, Johansen, Abrahamsen, Schüller, & Skeie, 2016; Veith & Reynolds, 2004; Xianghe, Pan, Peilong, Ismail, & Voorts, 2012). Therefore, it was of interest to compare the impact of CYS on the denaturation, separation and properties of the WPC and WPI samples. There was no detectable denaturation of whey proteins in the unheated samples regardless of the level of CYS added. Denaturation was only observed in samples that were heated, and the level of denaturation was dependent on the particular protein of interest (α-
lactalbumin or β-lactoglobulin), the level of added CYS and the duration of heating at 63 °C (Fig. 1). This is consistent with our previous investigation (Nguyen et al., 2018).
3.1.
Small scale experiments
Preliminary experiments were conducted to determine the conditions to use in subsequent larger scale experiments. In the first experiments, the whey protein solutions (10 and 15%, w/w) were heat-treated at 63 °C for 10 to 30 min with 0 to 20 mM of added CYS. For the WPC, all the β-lactoglobulin remained native, regardless of the CYS level, the holding time at 63 °C or the WPC concentration (Fig. 1A,B). In contrast, the level of denatured α-lactalbumin was dependent on the CYS level, the holding time at 63 °C and the WPC concentration. No denaturation of α-lactalbumin was observed in the heated sample with no CYS added. However, in samples with CYS added, there were higher levels of denaturation at the higher CYS concentrations, longer holding times, and at the higher WPC concentrations. For the 10% WPC solutions with 10 mM CYS, about 55%, 75% and 85% of the α-lactalbumin was denatured after heating for 10, 20 or 30 min respectively, with the levels increasing to 80, 90 and 95% for the 15% WPC solutions after 10, 20 or 30 min respectively (Fig. 1A,B). As with the WPC, no denaturation of β-lactoglobulin was observed for the WPI samples regardless of the WPI concentration, the holding time at 63 °C or the level of added CYS. Similarly, no denaturation of α-lactalbumin was observed in the heated samples without CYS added. However, the denaturation of α-lactalbumin was more extensive than for the WPC samples when CYS was added, and a similar behaviour was observed at both WPI concentrations. At 2 mM added CYS and 10 min heating, about 60% of the α-lactalbumin was denatured, and this increased to more than 80% at longer heating times or at the higher CYS
concentrations. For the WPI samples heated for 10 and 20 min, very low levels (≤ 2%) of native α-lactalbumin could be detected at CYS levels ≥ 7 mM, whereas for samples heated for 30 min, very low levels (≤ 2%) of native α-lactalbumin could be detected at CYS levels ≥ 5 mM (Fig. 1C,D). As the denaturation of α-lactalbumin in WPC at each CYS concentration was conducted at three holding times (600, 1200 and 1800 s), using equation 1 (with the assumption that the denaturation of α-lactalbumin followed first order reaction kinetics), the natural logarithm of the denaturation level of α-lactalbumin in the WPC solutions (Ct/C0, where C0 is the initial α-lactalbumin concentration and Ct is the α-lactalbumin concentration after heating for time (t)) was plotted against the holding time (t) in seconds (Fig. 2A,B). According to equation 1, the rate constants (k1) were obtained from the slopes of the straight lines (-k1). Interestingly, a linear relationship was observed when the rate constants were plotted against the log of the CYS concentration (Fig. 2E). The denaturation of α-lactalbumin in the WPI samples was more rapid than in the WPC samples. As some samples had very low levels (≤ 2%) of native α-lactalbumin remaining, these could not be reliably used in the kinetic evaluation. Consequently, only a limited number of the results were used for determining the rate constants, which reduces the reliability of this kinetic evaluation (Fig. 2C,D). Despite this, the obtained rate constants showed a linear increase with the log of the CYS concentrations, as was observed for the WPC samples (Fig. 2E). These results confirmed that, at each CYS concentration, the slowest rate of α-lactalbumin denaturation was for the 10% WPC sample and the highest rate of α-lactalbumin denaturation was for the 10% and 15% WPI samples (both similar), with the 15% WPC sample intermediate between these. = − . (1)
3.2.
Large scale experiments
From the results of the small-scale experiments, 10% WPC and WPI solutions with CYS concentrations of 0 and 10 mM and a heating temperature of 63 °C and a holding time of 20 min were used for the larger (10 L) scale experiments. After heating and cooling, the original unheated and the heated WPC and WPI samples were adjusted to pH 4.6 and centrifuged and the supernatants collected. The level of native α-lactalbumin and βlactoglobulin in the samples determined by microfluidic chip SDS PAGE. No α-lactalbumin or β-lactoglobulin was denatured in the samples heated in the absence of CYS, whereas in the sample with 10 mM CYS, virtually all the α-lactalbumin but very little β-lactoglobulin was denatured (Fig. 3), consistent with the results of the small-scale experiments. The supernatants were readjusted to pH 6.8 and spray dried to produce dried powders. A total of six powders were produced: WPC and WPI powders from the supernatants of the unheated samples (denoted WPCcontrol-10 and WPIcontrol-10 respectively), WPC and WPI powders from the supernatants of the heated samples (denoted WPCheated-10 and WPIheated-10 respectively), and WPC and WPI powders from the supernatants of heated samples with CYS added and thus enriched in β-lactoglobulin (denoted WPCβ-LG-10 and WPIβ-LG-10 respectively). The moisture, NPN, TN and calculated protein concentrations of the powders are shown in Table 1. The moisture ranged from 7.3 to 8.4%, which is higher than typical commercial powders, probably due to the milder dying conditions used. The total nitrogen levels of the WPC powders, both “as is” or on a dry basis were similar, as were those of the WPI powders; although the TN was slightly lower for the WPCβ-LG-10 and WPIβ-LG-10. As αlactalbumin has higher nitrogen levels than β-lactoglobulin (Grappin & Ribadeau-Dumas, 1992) its removal will lower the overall nitrogen content of the dried powder. In addition, due
to the removal of α-lactalbumin, the proportion of protein to non-protein components will decrease resulting in a lower nitrogen content of the dry powders. In these experiments the residual CYS was not removed from the supernatants prior to drying and therefore the nonprotein nitrogen contents were higher for WPCβ-LG-10 and WPIβ-LG-10 than for the other WPC and WPI samples. Attempts were made to re-disperse the α-lactalbumin-enriched pelleted material from the heated samples with CYS added; however, the pellet was firm and could not be fully re-dispersed and could not be spray dried. The WPC and WPI powders were reconstituted in water and analysed for protein content by HPLC, which gave a more detailed breakdown of the protein composition of each powder. The protein composition of the WPCcontrol-10 and WPCheated-10 samples were similar indicating that the heating had little effect on all proteins present, including the more heat labile IgG and BSA (Fig. 4). For the WPCβ-LG-10 sample, virtually no α-lactalbumin was present, and the BSA level was also markedly reduced compared with the unheated and heated control samples. Overall about 85% of the calculated protein content ((TNNPN)*6.38) was accounted for by this protein analysis, indicating some other protein species may be present in the WPC. For the WPIcontrol-10 and WPIheated-10 samples, β-lactoglobulin was the dominant protein with lower levels of α-lactalbumin, and only very low levels of the other whey proteins. For the WPIβ-LG-10 sample, high levels of β-lactoglobulin were present and only very low levels of α-lactalbumin and the other whey proteins were detected. About 95% of the calculated protein content ((TN-NPN)*6.38) was accounted for by this protein analysis, indicating only low levels of other protein species were present in the WPI. Skim milk samples and skim milk samples with 1% protein (based on (TNNPN)*6.38) from the WPC and WPI powders were heated at 80 °C for 30 min, cooled and then slowly acidified with GDL to form gels. The pH changes and the rheological properties of the samples were measured during acidification. The pH decline after addition of GDL
was very similar for all samples including the skim milk (Fig. 5), which indicates that the added WPC and WPI did not significantly modify the buffering of the milk. This is consistent with previous reports on acid gelation of skim milk samples fortified with various whey protein samples (Anema, 2018, 2019; Nguyen, Wong, Havea, Guyomarc’h, & Anema, 2013). Therefore, any differences in acid gelation are not due to pH differences during acidification. Skim milk produced an acid gel with a final stiffness (after 3 h of gelation) of about 190 Pa. Addition of the WPCcontrol-10 and WPCheated-10 powders to the milk samples increased the G’ to about 250Pa (Fig. 5A). When the WPCβ-LG-10 powder was added to milk before gelation, the final stiffness was substantially higher at about of 400 Pa (Fig. 5A), indicating a significant improvement in acid gelation properties compared with the standard WPCcontrol-10. The addition of 1% protein from WPI powders to skim milk prior to heat treatment and acid gelation caused a marked increase in the final G’, from 190 Pa in the gelled skim milk to about 550 Pa in the gelled skim milk with 1% added WPI. Unlike the WPC samples, there were only small differences in the final G’ between the WPIcontrol-10, WPIheated-10 and WPIβ-LG-10 powders (Fig. 5B). The sample with the WPIβ-LG-10 powder added was slightly higher (590Pa) than the other samples. The final G’ from the acid gels with added WPI were much higher than those from the skim milk with added WPC. A further set of experiments was conducted using 2% WPC. Samples had 5 mM CYS added before heating at 63 °C for 20 min. After heating, the pH was adjusted to 4.6 and the precipitate that formed was allowed to settle overnight. The supernatant was gently decanted from the sediment. Microfluidic chip SDS-PAGE was conducted on the supernatant and sediment, which showed that the supernatant was composed of β-lactoglobulin with no detectable α-lactalbumin, whereas the sediment was composed of both α-lactalbumin and βlactoglobulin (Fig. 6). The sediment sample was diafiltered with 5 equivalent volumes of water using a 0.1 µm microfiltration membrane. The retentate and permeate sample from the
sediment was analysed by microfluidic chip SDS-PAGE after each volume of water, and it was evident that the α-lactalbumin level in the retentate increased and the β-lactoglobulin level in the retentate decreased (Fig. 6) and was found in the permeate (result not shown) indicating that the β-lactoglobulin passed through the membrane whereas the (denatured) αlactalbumin did not pass through the membrane. The β-lactoglobulin enriched supernatant was diafiltered with 5 equivalent volumes of water using a 30 kDa ultrafiltration membrane. After diafiltration, the samples were adjusted to pH 6.8 and spray dried. The powder produced was enriched in β-lactoglobulin and denoted WPCβ-LG-2. It was difficult to spray dry the sediment sample as it regularly blocked the drier nozzle, but eventually enough powder was obtained to do some analysis. The powder from the sediment was enriched in denatured α-lactalbumin and denoted as WPCα-LA-2. The moisture, TN and NPN levels of the powders were determined (Table 1). The moisture levels of the WPCβ-LG-2and WPCα-LA-2 powders were lower than the previously prepared WPC powders. Due to the diafiltration, the excess CYS was removed therefore the NPN was lower, and, as the diafiltration removes lactose and other small molecules, the TN and calculated protein levels were higher than in the previous WPC samples. Skim milk was fortified with 1% protein from the WPCβ-LG-2 and WPCα-LA-2 powders and then slowly acidified with GDL to form gels, with the rheological properties followed during the acidification (Fig. 7). The skim milk with protein from WPCβ-LG-2 powder formed a gel with a final stiffness of about 450 Pa which was markedly higher than the gel obtained from skim milk alone (~190 Pa) and similar to that obtained with the WPCβ-LG-10 powder (Fig. 5A). In contrast, the skim milk sample with protein from the WPCα-LA-2 powder produced an acid gel with a final stiffness of about 120 Pa, which was lower than the gel from the skim milk alone and markedly lower than that from the skim milk with protein from the WPCβ-LG-2 powder.
4.
Discussion
At any given heating time at 63 °C, the level of denaturation of α-lactalbumin in the WPC and WPI samples increased as the CYS level increased (Fig. 1). The denaturation of αlactalbumin in skim milk and whey protein solutions is reported to follow pseudo-first order reaction kinetics (Anema & McKenna, 1996; Dannenberg & Kessler, 1988; Kessler & Beyer, 1991), thus an evaluation using first order kinetics was used to determine the rate constants and these were found to increase with the log of the CYS concentration (Fig. 2). The proposed mechanism for the effect on CYS on the denaturation of α-lactalbumin is that when the α-lactalbumin unfolds to a molten globule state at temperatures above about 61 °C, it exposes its disulphide bonds. In the absence of species with reactive free thiol groups, the α-lactalbumin will refold to its native conformation when cooled; however, in the presence of a reactive free thiol group, the thiol will initiate thiol-disulphide exchange reactions causing the α-lactalbumin to aggregate and thus unable to refold. For the denaturation/aggregation reactions of the whey proteins in milk and whey solutions, at temperatures below ~80 °C for α-lactalbumin and at temperature below ~90 °C for β-lactoglobulin, the unfolding of the protein is considered to be the rate determining step, with the subsequent aggregation reaction occurring rapidly. For α-lactalbumin at 63 °C, the addition of CYS is unlikely to change the rate of unfolding of the protein, but will increase the propensity of aggregation by initiating thiol-disulphide exchange reactions, allowing for irreversible aggregation to occur. Although only low levels of free thiols would be required to initiate the thiol-disulphide exchange reactions, due to the high pKa (~8.5) of the thiol group of free CYS (Netto et al., 2007) the concentration of the reactive thiolate group (S-) in CYS will be very low at the pH used in these experiments thus an increasing rate of aggregation
would be expected with increasing CYS concentrations until sufficient S- is introduced relative to the number of disulphide bonds (Figs. 1 and 2). Although α-lactalbumin is the predominant protein denatured in WPC and WPI when heated in the presence of CYS (Figs. 3 and 4), the minor whey protein BSA was also denatured under these heating conditions (Fig. 4). The denaturation temperature of BSA is lower than that of α-lactalbumin at about 62 °C (Paulsson et al., 1985; Ruegg et al., 1977), indicating that it is unfolding at the heating conditions used. However, the observation that it is not irreversibly denatured on heating indicates that the free thiol in BSA is not reactive at these low temperatures, although it probably is exposed and reactive at higher temperatures as BSA denaturation is irreversible when cycled between 20 and 110 °C (Ruegg et al., 1977). By precipitation and centrifugation (Figs. 3 and 4) or ultrafiltration/microfiltration with diafiltration (Fig. 6) and subsequent spray drying, it was possible to produce modified whey protein powders. Addition of the WPCcontrol-10 and WPCheated-10 powders to skim milk before heating and acidification increased the stiffness of the acid gels by about 30% (Fig. 5A). A similar, although larger increase in stiffness in acid gels was observed when a different type of WPC was added to skim milk at similar addition levels prior to heating and acidification (Lucey, Munro, & Singh, 1999). The results of this previous study are not directly comparable as the GDL level was lower and the WPC used was optimised for gelation and cultured applications (Fenwick & MacGibbon, 2014); however, these results demonstrate that the stiffness of acid gels is increased when whey protein concentrates are added to milk. When the WPIcontrol-10 and WPIheated-10 powders were added to the milk before heating and acidification the stiffness of the gels increased by about 250% (Fig. 5B), substantially more than when the WPC was added (Fig. 5A). Unlike WPC, which contains β-lactoglobulin, α-lactalbumin and GMP at significant levels, the WPI is composed predominantly of β-
lactoglobulin with a lower proportion of α-lactalbumin, and virtually no GMP (Fig. 4). As βlactoglobulin contributes more to the acid gel stiffness than α-lactalbumin (Graveland-Bikker & Anema, 2003), the much higher proportion of β-lactoglobulin in the WPI than the WPC would probably account for the larger increase in stiffness of the acid gels prepared from milk fortified with WPI. When the WPCβ-LG-10 powder was added to the milk before heating and acidification, a marked increase in acid gel stiffness was observed (Fig. 5A). This WPCβ-LG-10 powder contained residual CYS and previous studies have shown that the stiffness of acid gels prepared from skim milk could be markedly increased by the addition of thiol reagents (Nguyen et al., 2013). However, the level of residual CYS in the milk would be low (less than 1 mM) and unlikely to have a significant effect at these low concentrations as previous studies have shown that thiol reagents at these low concentrations had only small effects on the acid gelation properties of heated milk (Nguyen et al., 2013). In addition, a very similar increase in gel stiffness was obtained when the diafiltered WPCβ-LG-2 powder was used (Fig. 7), suggesting that the increase in stiffness was due to the removal of the α-lactalbumin and thereby increasing the proportion β-lactoglobulin in the sample. The acid gel containing the WPCα-LA-2 powder sample had a lower stiffness consistent with reports that show denatured/aggregated whey proteins have a detrimental effect on acid gel properties (Lucey, Tamehana, Singh, & Munro, 1998; Schorsch, Wilkins, Jones, & Norton, 2001). In contrast to the WPCβ-LG-10 and WPCβ-LG-2 powders, when the WPIβ-LG-10 powder was added to milk before heating and acidification, only a small increase in acid gel stiffness was observed (Figure 5B). The WPIcontrol-10 and WPIheated-10 powders have a high level of βlactoglobulin and a proportionally low level of α-lactalbumin (Fig. 4), and WPI addition to milk markedly increases acid gel stiffness compared with WPC (Fig. 5). Therefore, the
removal of the α-lactalbumin from the WPI appears to have only a small effect on the WPI performance in an acid gel system.
5.
Conclusion
Addition of CYS to concentrated (10 and 15%, w/w) WPC and WPI solutions before heating at 63 °C resulted in the denaturation of α-lactalbumin with β-lactoglobulin remaining native. The level of denaturation of α-lactalbumin increased with the level of added CYS at each heating time and with heating time at each level of added CYS. As α-lactalbumin unfolds at temperatures above 60 °C whereas β-lactoglobulin unfolds at temperatures above 70 °C, the CYS can initiate thiol-disulphide exchange reactions with the disulphide bonds of the unfolded α-lactalbumin at 63 °C producing intermolecular disulphide bonded aggregates, thus preferentially denaturing the α-lactalbumin. The denaturation of α-lactalbumin in the presence of CYS could be described as a (pseudo) first order reaction and the denaturation rate constant increased linearly with the log of the CYS concentration, indicating that the CYS accelerated the irreversible denaturation and aggregation of the α-lactalbumin. WPC solutions at 2% and 10% (w/w) and WPI solutions of 10% (w/w) that were heated in the presence of CYS could be separated into precipitates containing denatured αlactalbumin and solutions containing the remaining native whey proteins, in particular βlactoglobulin. These could be spray dried producing modified WPC and WPI powders that were either depleted in α-lactalbumin/enriched in β-lactoglobulin (WPCβ-LG-10, WPCβ-LG-2, and WPIβ-LG-10) or enriched in denatured α-lactalbumin (WPCα-LA-2). Although some difficulty was encountered drying the WPCα-LA-2 due to nozzle blockages, these results indicate that WPC and WPI products with modified protein compositions could be prepared by preferential denaturation and separation of α-lactalbumin from the other whey proteins.
Surprisingly, when the WPIcontrol-10, WPIheated-10, and WPIβ-LG-10 were added to skim milk, heated and acidified, the gels formed had similar properties. This is probably due to the WPIcontrol-10 and WPIheated-10 having high levels of β-lactoglobulin and markedly increasing acid gel stiffness so that minimal further improvement was achieved by increasing the βlactoglobulin level in the WPIβ-LG-10. In contrast, markedly different gel properties were obtained when the WPCcontrol-10, the WPIheated-10, the WPCβ-LG-10, the WPCβ-LG-2, or the WPCαLA-2
were added to the skim milk before heating and acidification, with the WPCα-LA-2
decreasing the acid gel stiffness and the WPCβ-LG-10 and WPCβ-LG-2 increasing the acid gel stiffness compared with the WPCcontrol-10 and WPCheated-10, which were similar. The WPCcontrol-10 and WPCheated-10 have lower levels of β-lactoglobulin than WPIcontrol-10 and WPIheated-10, and only causes moderate increases in acid gel stiffness, thus increasing the βlactoglobulin level in the WPCβ-LG-10 and WPCβ-LG-2 powders may have a more marked effect on the acid gels than for the WPI samples. As the α-lactalbumin in the WPCα-LA-2 is already denatured and aggregated, it probably does not contribute to the acid gel structure and thus a gel with decreased stiffness is obtained. Overall these results indicate that WPC samples with modified functional properties could be prepared by the preferential denaturation and separation of α-lactalbumin through reaction with CYS.
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Figure legends
Fig. 1. Effect of CYS concentration on the level of native β-lactoglobulin ( , ▲, lactalbumin ( , ▲,
) and α-
) in 10% WPC (A), 15% WPC (B), 10% WPI (C) or 15% WPI (D)
solutions heated at 63 °C for 10 ( ,
), 20 (▲, ▲) or 30 ( ,
) min. Native protein levels
were determined by traditional SDS-PAGE.
Fig. 2. Denaturation of α-lactalbumin WPC (A, B) and WPI (C, D) solutions as a first order reaction. A, 10% WPC; B, 15% WPC; C, 10% WPI; D, 15% WPI with 2 mM ( ), 5 mM ( ), 7 mM (▲), 10 mM (▲) or 20 mM ( ) added CYS. Samples were heated at 63 °C for different times. For the WPC samples (A, B) all data were used for obtaining rate constants; for the WPI samples (C, D) only samples that the lines pass through were used for obtaining rate constants (see text for explanation). E: rate constants plotted against CYS concentration for 10% WPC ( ), 15% WPC ( ), 10% WPI (▲) and 15% WPI (▲).
Fig. 3. Level of native β-lactoglobulin (red bars) and α-lactalbumin (blue bars) in unheated and heated (63 °C, 20 min) 10% WPC and 10% WPI samples. Native protein levels were determined by microfluidic chip SDS PAGE.
Fig. 4. Protein composition of spray dried WPC and WPI powders from unheated and heated (63 °C, 20 min) samples as determined by reversed-phase HPLC. Red bars: β-lactoglobulin, blue bars: α-lactalbumin, green bars: glycomacropeptide, black bars: BSA, pink bars: PP5, teal bars: IgG.
Fig. 5. Gelation curves showing the change in stiffness and pH with time after 2% GDL was added to heated (80 °C, 30 min) milk samples. A: Acid gels were prepared from skim milk ( , ▲), skim milk with 1% protein from WPCcontrol-10 ( , ▼), WPCheated-10 (▼) or WPCβ-LG10
(▲). B: Acid gels were prepared from skim milk ( , ▲), skim milk with 1% protein from
WPIcontrol-10 ( , ▼), WPIheated-10 (▼) or WPIβ-LG-10 (▲).
Fig. 6. Protein composition of the supernatants, sediments, and sediment retentates during diafiltration with 1 (R1), 2 (R2), 3 (R3), 4 (R4), and 5 (R5) equivalent volumes of water. Supernatants and sediments were obtained from 2% WPC heated in the presence of 5 mM of CYS. Red bars: β-lactoglobulin, blue bars: α-lactalbumin. Native protein levels were determined by microfluidic chip SDS-PAGE.
Fig. 7. Gelation curves showing the change in stiffness with time after 2% GDL was added to heated (80 °C, 30 min) skim milk ( ), skim milk with 1% protein from WPCβ-LG-2 ( ), or skim milk with 1% protein from WPCα-LA-2 (▼).
1
Table 1
2
Moisture, total nitrogen, non-protein nitrogen and calculated protein concentrations of WPC and WPI powders. a Powder descriptor
Powder sample starting material
Moisture (%, w/w)
Total nitrogen (%, w/w)
Non-protein nitrogen (%, w/w)
Protein (TN-NPN)*6.38 (%, w/w)
WPCcontrol-10
10% WPC unheated
7.7
11.69 (12.67)
0.657 (0.712)
70.39 (76.26)
WPCheated-10
10% WPC heated
7.3
11.65 (12.57)
0.897 (0.968)
68.60 (74.01)
WPCβ-LG-10
10% WPC/10 mM CYS supernatant heated
8.2
11.16 (12.16)
1.26 (1.373)
63.16 (68.80)
WPIcontrol-10
10% WPI unheated
8.3
13.81 (15.06)
0.098 (0.107)
87.48 (95.40)
WPIheated-10
10% WPI heated
7.4
13.93 (15.04)
0.086 (0.093)
88.32 (95.38)
WPIβ-LG-10
10% WPI/10 mM CYS supernatant heated
8.4
13.57 (14.81)
0.330 (0.360)
84.47 (92.22)
WPCβ-LG-2
2% WPC/5 mM CYS heated supernatant
4.8
13.61 (14.30)
0.356 (0.374)
84.56 (88.82)
WPCα-LA-2
2% WPC/5 mM CYS heated retentate
2.8
12.23 (12.58)
0.213 (0.219)
76.67 (78.88)
3 4
a
Total nitrogen, non-protein nitrogen and calculated protein concentrations presented on an as is basis with values on a dry basis in parentheses.
1
100 80 60
Native protein (%)
40 20 0 100
A
B
C
D
80 60 40 20 0
0
5
10
15
20
0
Cysteine added (mM)
Figure 1
5
10
15
20
0
A
B
C
D
-1 -2
ln(Ct/C0)
-3 -4 0 -1 -2 -3 -4 -5 -6 0
500
1000
1500
0
500
1000
Rate constant (k1 * 1000)
Time (seconds)
E
6
4
2
0.2
0.4
0.6
0.8
1.0
log ([Cysteine])
Figure 2
1.2
1.4
1500
Figure 3
Intensity (arbitrary units) 600
Heated WPI with CYS
500
Heated WPI
400
Unheated WPI
300
Heated WPC with CYS
200
Heated WPC
100
0
Unheated WPC
Figure 4
-1
mg protein. g powder
Heated WPI Heated WPI with CYS
800
Unheated WPI
600
Heated WPC with CYS
400
Heated WPC
200
0
Unheated WPC
A 400
6.5
300
6.0 5.5 5.0
100 4.5 0 600
pH
Stiffness (G' in Pa)
200
B 6.5
500
6.0 400
5.5
300 200
5.0
100
4.5
0
0
50
100
Time (min)
Figure 5
150
Figure 6
Protein composition (%)
R4 R5
100
R3
80
R2
60
R1
40
Sediment
20
0
supernatant
Stiffness (G' in Pa)
400 300 200 100 0 0
50
100
Time (min)
Figure 7
150