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Properties of whey protein concentrate powders obtained by spray drying of sweet, salty and acid whey under varying storage conditions
Accepted Manuscript Properties of whey protein concentrate powders obtained by spray drying of sweet, salty and acid whey under varying storage conditions Manjula Nishanthi, Jayani Chandrapala, Todor Vasiljevic PII:
S0260-8774(17)30278-9
DOI:
10.1016/j.jfoodeng.2017.06.032
Reference:
JFOE 8937
To appear in:
Journal of Food Engineering
Please cite this article as: Manjula Nishanthi, Jayani Chandrapala, Todor Vasiljevic, Properties of whey protein concentrate powders obtained by spray drying of sweet, salty and acid whey under varying storage conditions, Journal of Food Engineering (2017), doi: 10.1016/j.jfoodeng.2017.06.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Properties of whey protein concentrate powders obtained by spray drying of sweet,
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salty and acid whey under varying storage conditions
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Manjula Nishanthi1, Jayani Chandrapala1,2, Todor Vasiljevic1*
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University, Werribee campus, Victoria 3030, Australia
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Advanced Food Systems Research Unit, College of Health and Biomedicine, Victoria
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School of Science, RMIT University, Bundoora, Victoria 3083, Australia
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*Corresponding Author:
Tel: +61 3 9919 8062
Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract
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Changes of secondary structure and protein interactions of whey protein (WPs) present in
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native, sweet, acid and salty-WPCs were analyzed following storage at 4, 25 or 45°C and 22
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or 33% relative humidity for a period of 90-days. WPs aggregated predominantly through
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covalent crosslinking, achieving maximum at 45 °C and 33% RH. Greater participation of β-
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lactoglobulin in covalent crosslinking was evident in all WPCs, while that of α-lactalbumin
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was significantly (p<0.05) high in acid-WPC only. Reaction order of β-LG denaturation in
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acid and salty-WPCs was approximately 2 while approximately 1 in native and sweet-WPCs.
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Activation energy was significantly (p<0.05) higher in native and sweet-WPCs, with
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averages recorded as 97 and 49.8 kJ mol-1, respectively, than that in acid and salty-WPCs with
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averages of 27.5 and 33.8 kJ mol-1, respectively, mainly attributed to inherently high
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concentrations of lactic acid and salts in these WPCs.
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Key word: Whey protein concentrate, protein aggregation, covalent interactions, acid whey,
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salty whey
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ACCEPTED MANUSCRIPT Introduction:
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Whey protein concentrates (WPCs) are an important protein source used in many food
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systems due to their excellent functional characteristics such as emulsification, gelling and
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foaming. Dairy industry has been successful in utilizing the sweet whey waste stream for
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manufacturing of various WPCs. However, the industry has not been very successful in
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utilizing acid and salty whey in an equivalent way to sweet whey, mainly due to their
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composition-induced processing obstacles such as high acidity and salinity, respectively.
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Moreover, the powders produced from these two streams form clumps and sticky deposits
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during storage. A recent study by Nishanthi et al. (2017a) showed that the particle surface of
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salty-WPC was hydrophobic mainly due to high surface fat content, while that of acid-WPC
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was characterized by both hydrophobic and hydrophilic nature due to presence of elevated
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protein concentration. Therefore, these two WPCs possess a high potential to be used as
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functional ingredients in food systems. Thus, a thorough fundamental understanding on the
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stability of proteins present in these respective WPCs as affected by storage is a necessity in
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evaluating the potential of using these two particular WPCs.
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Storage conditions induce changes in protein conformation at a molecular level, which in turn
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may affect functional properties of the protein. Intermolecular covalent and non-covalent
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interactions occur between whey proteins (WPs), resulting in protein unfolding and
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denaturation, eventually leading to reversible and irreversible aggregation. As a result, a
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proportion of native WPs decreases after 3 months of storage at 40 °C with a concomitant
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increase in proportion of denatured proteins. These changes were clearly temperature
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dependent as aforementioned changes were not noticed at 4 °C or 20 °C up to 15 months of
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storage (Norwood et al., 2016). At elevated storage temperatures, flexibility and mobility of
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WPs increases, resulting in partial loss of their tertiary structure (Norwood et al., 2016). WPC
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was found to have increased surface hydrophobicity and less exposed amino acids on the
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ACCEPTED MANUSCRIPT particle surfaces after storage at 60 °C and 22 % RH for one month (Burgain et al., 2016). In
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addition to denaturation, WPs can be subjected to lactosylation during storage. While studies
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on lactosylation of WPC powders are lacking, Le et al. (2012) reported that six out of twelve
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lysine residues of α-lactalbumin (α-LA) would lactosylate during storage of milk protein
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concentrates up to 12 weeks at 40 °C and 84 % RH, which indicates that similar changes
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could be expected in WPC, especially those containing greater lactose content. These
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structural modifications in WPs could subsequently affect their functional properties. For
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instance, formation of insoluble aggregates in response to elevated storage temperatures
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reduced the solubility of WPC (Zhou et al., 2008) affecting several functional characteristics
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such as emulsification, gelation and foaming.
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So far, the studies mainly focused on WPC produced from the sweet whey and no study thus
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far has established physico-chemical properties of WPs during storage of WPCs produced
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from acid or salty whey, which were identified as potentially effective functional ingredients
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(Nishanthi et al., 2017a). Therefore, the main aim of the current study was to establish
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conformational and surface interactional changes of WPs present in sweet, acid and salty
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WPCs under three different storage temperatures (4, 25, 45 °C) and two relative humidities
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(RH) (22% and 33%) over a storage period of 90 days. Native-WPC was used as the
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reference sample.
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2. Methodology
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2.1 Sample preparation
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Four WPCs, native-WPC, sweet-WPC, acid-WPC and salty-WPC were manufactured in our
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previous study (Nishanthi et al., 2017a) with the composition shown in Table 1 and used
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during storage trial in the current study. WPCs were first completely dehydrated by placing
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ACCEPTED MANUSCRIPT appropriate quantity of samples in vacuum desiccators over phosphorous pentoxide at room
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temperature (25°C) for 4 weeks.
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2.2 Storage trial
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Approximately 3 g of each WPC was placed in standard desiccators (with a porcelain disc
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size of 250 mm) with a controlled RH (22 and 33%), sealed and placed in temperature
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controlled cabinets at three temperatures (4, 25 and 45°C). The equilibrium RH in each
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desiccator was maintained using saturated salt slurries containing potassium acetate (22 ±
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2%) or magnesium chloride (33 ± 1%). At 4, 25 and 45 °C, magnesium chloride maintained
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RH of 34, 33 and 32%, while potassium acetate maintained RH of 24, 23 and 23%,
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respectively. Five desiccators were prepared for each temperature/humidity combination as
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sampling was conducted at 5 time points representing 0, 14, 30, 60 and 90 days of storage. A
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pre-trial test was conducted to estimate the equilibrium time and identify the first sampling
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point. Several samples from each WPC were stored in standard desiccators with saturated salt
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slurries of magnesium chloride and potassium acetate. After one week of storage, one sample
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from each WPC was tested for the water activity using a water activity meter (Aqualab CX-
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3T, Labcell, Hampshire, United Kingdom) at 25°C each day. The four types of WPCs reach
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to the water activity of 0.22 and 0.33 by one week and four days. A stabilised water activity
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level could be achieved within two weeks. Therefore, the first sampling (0 day) was
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conducted after allowing 2-weeks equilibrium period from the initiation date of the storage
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trial. After opening desiccators at a required time point, the analysis was carried out within 3
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days.
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2.3 Fourier Transform Infrared Spectroscopy (FTIR)
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The FTIR spectra of WPCs were obtained in the range of 4000 - 600 cm−1 using the
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PerkinElmer Frontier FTIR spectrometer (PerkinElmer, MA, USA) as described previously
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(Nishanthi et al., 2017b). The spectra were vector normalized, smoothened and deconvoluted
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ACCEPTED MANUSCRIPT with the aid of IR Solution software (Shimadzu Corporation, Kyoto, Japan), in order to
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recognize the corresponding peaks within the amide I region between 1600 and 1700 cm−1.
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2.4 Sodium Dodecyl Sulphate Gel Electrophoresis (SDS – PAGE)
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In order to establish non-covalent and covalent interactions, Sodium Dodecyl Sulphate (SDS)
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polyacrylamide gel electrophoresis (PAGE) was performed under reducing (SDSR) and non-
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reducing (SDSNR) conditions using freshly casted gels with 30% (w/v) acrylamide and 10%
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(w/v) SDS (Nishanthi et al., 2017b). Samples were reduced with addition of β-
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mercaptoethanol and subsequent heating at 100 °C for 5 min.
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2.5 Kinetics analysis
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The rate (1) and the Arrhenius equations (2) were used to calculate the kinetic parameters
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involved in disappearance of native WPs during storage of WPCs (Oldfield et al., 2005).
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− = (1)
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Where:
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C = Protein concentration (g kg-1)
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t = time (days)
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kn = rate constant ((g kg-1)(1-n) day-1)
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n = reaction order
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= (2)
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Where:
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k0 = pre-exponential term ((g kg-1)(1-n) day-1)
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Ea = activation energy (kJ mol-1)
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R = universal Gas Constant (8.314 J mol-1 K-1)
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T = temperature (K)
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2.6 Statistical analysis
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The experiments were organized in a randomized split plot blocked design with 3 main
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factors: a type of WPC as the main plot, and temperature and RH as subplots. The
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of the kinetic and compositional analysis were statistically analyzed using the General Linear
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Model (GLM) and ANOVA procedure, respectively. The level of significance was preset at
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p<0.05.
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3. Results and discussion:
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3.1 Secondary structural changes during storage through FTIR
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Significant and complex intermolecular interactions occur among WPs in response to storage
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conditions and associated compositional changes of examined WPCs. These changes affect
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the conformation of secondary structure motifs such as α-helices, β-sheets, β-turns and
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random coils in these proteins as shown in Figures 1 to 4.
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At the beginning of the storage trial, WPs present in native-WPC were characterized as β-
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sheet rich proteins due to two prominent peaks observed in the regions of 1628-1629 cm-1
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and 1693 cm-1 (Figure 1). However, other secondary structures, such as α-helixes, random
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peptides and β-turn were largely missing at 4 °C, but appeared with low intensities at 25 and
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45 °C, irrespective of RH. In addition to the native moieties, β-sheet cross linking (1682 cm-1
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and 1615 cm-1) observed at the beginning of storage can be attributed to the thermal impacts
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associated with pasteurization and spray drying applied during processing of this WPC.
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During initial stages of the storage (14-30 days), secondary conformations did not change
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substantially. However, after 60 days of storage WPs underwent several major structural
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changes. These changes were more pronounced at 33% RH as compared to that at 22% RH,
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but were independent of storage temperature. Firstly, complete distortion of α-helixes
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occurred under all storage conditions, which in turn increased the intensity of randomly
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arranged peptides (1644 cm-1). In presence of high amount of moisture (33 % RH),
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hydrophilic amino acid residues of α-helix form hydrogen bonds with water molecules, thus
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disturbing its intermolecular interactions. As a result of α-helical stretching, intensity of β-
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ACCEPTED MANUSCRIPT turns (1660 cm-1) increased attributed to the broadening of β-turn between β-strand I and H in
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β-lactoglobulin (β-LG). Secondly, widening of peaks attributed to β-sheets, observed
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prominently at 33% RH, suggests a possible intermolecular cross linking in the presence of
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elevated moisture content. Concomitantly, intensity of anti-parallel β-sheets (Lefèvre and
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Subirade, 1999) (1691 cm-1) increased, suggesting a dimerization or polymerization, moving
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high number of β-sheets to the molecular interior. Thirdly, greater exposure of amino acid
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(AA) side chain residues (1610 cm-1) (Lefèvre and Subirade, 1999) were observed mainly at
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33% RH. The random coils formed by stretching α-helixes may be small with high exposed
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surface area, which subsequently expose a high number of AA residues. Moreover, protein
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hydration due to greater relative humidity leads to movement of hydrophilic AA residues to
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the molecular exterior. At the end of the storage (90 days), the peaks representing β-turns and
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random coils widened and disappeared suggesting a possible aggregation that destructed
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native monomeric and dimeric forms of proteins. This loss of secondary structures was
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especially magnified in the samples stored at 45 °C and 33% RH. Further, the exposed side
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chain residues disappeared under all storage conditions. Interestingly, a greater content of β-
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sheets remained unaffected, suggesting the preservation of the calyx of β-LG.
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Sweet-WPC mainly contained intramolecular β-sheets (1630 and 1693 cm-1) and
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intermolecular β-sheets (1615 cm-1) at the start of the storage (Figure 2). Interlinking of β-
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sheets observed at the beginning of the storage suggests an impact of thermal treatments
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associated with the manufacturing process of sweet-WPC (Nishanthi et al., 2017a). Similar to
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native-WPC, initial storage period (14-30 days) did not change the profile of the secondary
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structure of sweet-WPC to a great extent for most storage conditions, although random coils,
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α-helixes and β-turns appeared in the samples stored at 33% RH. These changes can be
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attributed to the protein hydration, leading acquisition of some native attributes. Furthermore,
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a greater exposure of side chain AA residues was observed (1611 cm-1) at 22% RH. Similar
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ACCEPTED MANUSCRIPT to native-WPC, substantial structural changes were noticed after 60 days of storage.
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Irrespective of storage conditions, β-sheets lost their conformational stability, partially
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forming cross linked β-sheets (1680 cm-1). Strong modifications were seen in β-sheets
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indicated by appearance of two peaks (1623 and 1634 cm-1), which can be attributed to the
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disruption of intramolecular hydrogen bonds, leading to formation of new stronger
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intermolecular hydrogen bonds (Ngarize et al., 2004). This modification was especially
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prominent at 25 and 45 °C. Monomer-dimer equilibrium of β-LG molecules (Lefèvre and
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Subirade, 1999) has shifted towards the dimeric form, resulting in strongly bonded β-strand
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structures. Dimer formation, in parallel, leads to a high number of deeply buried β-type
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structures (1691 cm-1). Moreover, dimer formation takes place at expense of intramolecular
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α-helix, subsequently, resulting in high number of random coils. The α-helix stretching into
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random coils was high at 25 and 45 °C, while independent of RH. Therefore, the dimerization
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which was established in SWP after 60 days was a result of storage temperature. By the end
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of storage, secondary structures other than β-sheets completely disappeared in the samples
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stored at 25 and 45 °C. Furthermore, aging of sweet-WPC after 60 days induced a
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conformational change to the calyx of β-LG indicated by a shift in peak at 1691 cm-1 to a
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peak with low intensity at 1694 cm-1. This change in turn may lead to the exposure of reactive
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sites for covalent and hydrophobic interactions.
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In acid-WPC, β-sheets (1620 – 1635 cm-1) were largely absent at the beginning of the storage
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(Figure 3). Simultaneously, aggregation between anti-parallel β-sheets (1682 cm-1) was
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prominent at 22 % RH and 45 °C, involving mainly β-strands located on the surface, as
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opposed to those in deep areas of β-LG (1691 cm-1), which remained intact. Other secondary
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structures, such as α-helixes, β-turns and random coils were present at a low intensity,
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regardless of the storage conditions. By 14 days of storage, additional exposure of β-sheets
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(1621 cm-1) occurred suggesting a partial unfolding of β-LG in response to lactic acid (LA)-
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ACCEPTED MANUSCRIPT induced WP alterations (Nishanthi et al., 2017b). Unfolding of β-sheets was more prominent
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at 33% RH as compared to that at 22% RH. Deprotonation of LA occurs rapidly in the
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presence of moisture due to its low acid dissociation constant (pKa), which in turn decreases
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the negative surface charge density of the proteins promoting attractive forces, inducing
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protein aggregation driven by van der Waals and electrostatic interactions (Israelachvili,
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2011). After 60 days of storage, three significant modifications were noticed. Firstly, α-
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helixes disappeared completely forming non-native random structures under all storage
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conditions. Secondly, conformation of β-sheets changed substantially, converting into
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distinctive two peaks in the region of 1620 – 1638 cm-1, implying a dimerization of β-LG. In
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addition, under all storage conditions the intensity of deeply located β-sheets (1693 cm-1) was
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doubled. In presence of LA, β-LG formed dimers, relocating more of β-sheets to the inner
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side of the molecule. Thirdly, side chain amino acid residues (1610 cm-1) were greatly
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exposed, especially at 22% RH as a result of β-sheet modifications and α-helix disruption.
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Towards the end of the storage (90 day), a greater loss of secondary structures was observed,
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especially in the samples stored at 25 and 45 °C under 22% RH. A greater loss of random
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coils and dimeric forms of β-LG suggested a severe aggregation.
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In contrast to other WPCs, secondary structures of salty-WPC (Figure 4) showed high
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sensitivity to storage conditions during the first days of storage. For instance, proportions of
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β-sheets (1626, 1636 and 1693 cm-1), random peptides (1648 cm-1) and β-turns (1660 cm-1)
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clearly declined as temperature and humidity increased. In parallel, a number of deeply
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located β-sheets (1693 cm-1) descended with increase in temperature, denoting an exposure of
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the β-LG core. However, the temperature-dependent reactivity of β-LG did not increase the
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proportion of cross linked β-sheets (1616 and 1680 cm-1). Due to osmotic pressure caused by
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Na+ in the medium, β-sheets did not interlink, instead unfolded, forming unordered structures,
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as apparent by intensity of high random coil. A dimeric form of β-LG was also observed at 4
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ACCEPTED MANUSCRIPT and 25 °C (1620 – 1636 cm-1), as opposed to monomeric β-LG at 45 °C. Other secondary
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motifs were also greatly absent at 45 °C. During further storage (14-30 days), monomeric β-
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LG dominated (Lefèvre and Subirade, 1999) under all storage conditions, suggesting that
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native-dimers dissociated with aging. With increase in temperature, concomitant reduction of
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non-native monomers and enhanced aggregation driven by crosslinking of β-sheets (1616 cm-
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storage, β-sheets reappeared in the region of 1620 – 1638 cm-1, whereas the peak intensity
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increased with the storage temperature. Interestingly, the region denoted β-sheets (1620 –
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1636 cm-1) consisted of two peaks at 25 and 45 °C, denoting dimeric forms of β-LG, while it
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was just a single peak at 4 °C, attributed to monomeric β-LG. Dimeric β-LG formation in
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turn increased the number of intermolecular β-sheets (1680 cm-1), resulting in substantially
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greater proportion of deeply located β-sheets (1691 cm-1) and high number of random coils
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and β-turns. Up to 60 days, the observed structural changes were independent of the
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humidity. However, towards the end of storage (90 day), humidity influenced the interactions
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among β-sheets. High occurrence of intermolecular hydrogen bonds prominent at 22% RH
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suggested a high occurrence of β-LG dimers (1620 – 1636 cm-1), while intramolecular
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hydrogen bonds predominated at 33 % RH, suggesting more of monomeric form of β-LG.
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The prolonged exposure to high humidity reduced the protein-protein interactions, instead
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allowing protein hydration and formation of near-native protein monomers. This assumption
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is also supported by the occurrence of less cross linked β-sheets (1615 cm-1) at 33% RH.
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3.2 Changes in protein interactions and aggregation during storage
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At the beginning of the storage trial, aggregates (Mw≥250 kDa) covalently linked via
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disulphide bridges were observed in native-WPC (Figure 5 and 6) accompanied with
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subsequent disappearance of β-LG directly related to temperature, humidity and storage time.
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Intensity of α-LA bands on the other hand remained approximately consistent during initial
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involved in the overall aggregation process at a later stage. It is known that α-LA contains
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only four disulphide bonds positioned at Cys6-Cys120, Cys28-Cys11, Cys61-Cys77 and Cys73-
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Cys91 (McSweeney, 2013), but no free thiol groups. In addition, the holo-form of α-LA,
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which is present abundantly in native-WPC, provides a high thermal stability (Boye and Alli,
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2000). For these reasons, involvement of α-LA in overall aggregation is limited. Small
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amount of caseins was present in this WPC, which appeared to be involved in disulphide
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linking over the storage period, with the greatest extent at 45 °C and 33% RH.
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Thiol/disulphide exchange reaction usually occurs between Cys160 of β-LG and free thiols of
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Cys11 and Cys88 of к-casein (Creamer et al., 2004) forming an aggregate with a minimum
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Mw of ~ 37 kDa (Figure 5). Similar to other proteins, bovine serum albumin (BSA) was also
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involved in aggregation. However, its involvement was clearly storage time dependent, while
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other main factors had no effect. BSA forms disulphide crosslinks with β-LG (Matsudomi et
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al., 1994) due to a free thiol group at Cys34. In addition to disulphide bridging, WPs in native-
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WPC participated in non-reducible aggregation (Figure 7), likely due to lactosylation of
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lysine residues in these proteins. Compared to other WPCs, lactosylation was less prominent
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in this WPC without a clear association with storage temperature and time, which could have
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been caused by a very low amount of lactose.
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Sweet-WPC samples exhibited a similar behavior to that of native-WPC presented by clear
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formation of protein aggregates covalently linked by disulphide bridging over the overall
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storage period (Figure 5 and 6), again achieving maximum aggregation at 45 °C and 33%
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RH. In parallel to the aggregate formation, a rate of β-LG disappearance was the greatest
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under the same conditions (Table 2), suggesting that β-LG was the main protein involved in
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aggregation. Conversely, only minor involvement of α-LA in aggregation was observed
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under all storage conditions. No involvement of BSA was evident in sweet-WPC under all
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ACCEPTED MANUSCRIPT storage conditions, although comparatively a higher concentration of BSA was present in
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comparison with other WPCs. In contrast, caseins were involved in disulphide crosslinking,
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but a clear association with storage conditions and time could not be established. In addition
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to aggregates linked by disulphide bonds, two distinct bands representing non-disulphide
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linked aggregates appeared with approximate Mw of 71-72 kDa and ≥250 kDa (Figure 6).
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Compared to native-WPC, these aggregates were prominent in sweet-WPC, showing a
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gradual increase with rise in the storage temperature, humidity and time. Sweet-WPC
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contained 2.9% of lactose, which might have led to lactosylation of lysine segments of β-LG.
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Among these, Lys8 located at N terminal, Lys141 located at C terminal end and Lys138 located
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in 3 turn α-helix are easily accessible, therefore, likely the primary sites of the reaction.
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In acid-WPC, disulphide linked aggregates gradually increased throughout the storage with
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subsequent disappearance of β-LG A, β-LG B and α-LA (Figure 5 and 6). Rapid involvement
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of β-LG A was observed in aggregation, compared to β-LG B (Oldfield et al., 1998), mainly
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due to disulphide bridging via Val118 in β-LA A as compared to Ala118 in β-LG B, located in
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β-strand H, creating a less favorably packed core (McSweeney, 2013). This in turn increases
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the flexibility of β-LG A and exposes the free thiol group. Compared to other WPCs,
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involvement of α-LA in protein aggregation was greater in acid-WPC. Less amount of water
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might have restricted the availability of Ca2+ to the binding site of α-LA, preventing protein
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stabilization and thus resulting in prevalence of less stable apo-α-LA. The contribution of
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caseins in disulphide bonding was apparent only at 45 °C and 33% RH conditions, although a
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significantly higher amount of caseins was present in acid-WPC than in other WPCs
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(Nishanthi et al., 2017a). A diffuse region was observed in the region between caseins and
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BSA (Mw~50 kDa) in the samples stored at 45 °C and two RHs under reducing PAGE
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conditions (Figure 6), suggesting a possible aggregation between caseins and β-LG and/or α-
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LA via non-reducible covalent bonds. In addition to reducible aggregates, non-reducible
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ACCEPTED MANUSCRIPT aggregates likely created by lactosylation with Mw>250 kDa were observed in acid-WPC
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under all storage condition (Figure 6). The extent of aggregation was clearly temperature and
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RH dependent (Figure 7) and predominantly involved α-LA, as evidenced by the reaction
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rates (Table 3). Out of twelve lysine residues in α-LA, maximum of 6 residues (Lys24, Lys112,
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Lys117, Lys127, Lys133 and Lys141) are lactosylated easily (Le et al., 2012).
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Similar to other WPCs, salty-WPC contained disulphide linked protein aggregates with
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Mw>250 kDa (Figure 5 and 6). The aggregation increased with the storage time, achieving
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the maximum at 45 °C and 33% RH. Both β-LG A and β-LG B participated in disulphide
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bonding, however, β-LG A completely disappeared after 14 days, suggesting a high reaction
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rate (Oldfield et al., 1998). Involvement of α-LA in disulphide bonding was observed with
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extent clearly dependent of the storage temperature and time with no clear association with
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humidity. Thus, thiol/disulphide interchange reactions between β-LG and α-LA could be the
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main possible aggregation pathway. In absence of a reactive thiol group, thiol/disulphide
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interchange reactions occur through the disulphide bonds of Cys6-Cys120 and Cys28-Cys111
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located on the surface of α-LA, which form new disulphide bonds with Cys66 and Cys160
324
located on the surface of β-LG. Compared to other WPCs, salty-WPC contained a high
325
concentration of lactoferrin (LF) (Mw~87 kDa), which did not participate in covalent
326
aggregation under all storage conditions. On the other hand, BSA was greatly involved
327
concomitantly with the rise in storage temperature, humidity and time. Due to a free thiol
328
group at Cys34 and seventeen disulphide bonds in its primary structure, BSA participates in
329
both disulphide bridging and thiol/disulphide interchange reactions with β-LG (Matsudomi et
330
al., 1994) and α-LA (Matsudomi et al., 1993). Similar to sweet-WPC, salty-WPC contained
331
aggregates linked via covalent non-disulphide bonds in the region of BSA-LF with
332
approximate Mw of 71-72 kDa. Furthermore, the amount of large aggregates (Mw≥250 kDa)
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ACCEPTED MANUSCRIPT formed via lactosylation increased in parallel with the storage temperature, humidity and
334
time.
335
In four WPCs, type of proteins and interactions involved in protein aggregation vary with the
336
type of powder, storage conditions and time. The main protein involved in disulphide and
337
non-disulphide covalent aggregation was β-LG. Therefore, kinetic parameters of β-LG were
338
estimated to thoroughly understand the differences in behavior of β-LG in these WPCs.
339
3.3. Kinetics of β-LG disappearance
340
Kinetic parameters of β-LG disappearance in different WPCs during storage are presented in
341
Table 2. Disappearance of β-LG follows the common reaction kinetic principles. A
342
substantial reaction occurs if an adequate number of β-LG molecules with sufficient
343
activation energy (Eo) collide at an adequate frequency while maintaining specified molecular
344
orientation to form disulphide or other covalent bonds. Rate of disappearance of β-LG (Rβ-LG)
345
in native-WPC and sweet-WPC was directly related to the storage temperature and humidity,
346
being the greatest at 45 °C and 33% RH. Elevated temperature increases the number of
347
reactive β-LG molecules with exposed free thiol group of Cys121. High RH increases water
348
activity thus favors thermal energy transition and molecular motions, facilitating formation of
349
reactive monomers. In contrast, Rβ-LG of salty-WPC and acid-WPC decreases as RH
350
increases. This observation suggests a greater interference of compositional constituents
351
present in these two WPCs on Rβ-LG. As a result of the low pH in presence of high
352
concentration of LA in acid-WPC (Nishanthi et al., 2017a), dimerization of β-LG and
353
formation of octamers (Townend and Timasheff, 1960) occurs as evidenced by the FTIR
354
spectra and SDSNR PAGE patterns. Due to the large structure of octamer, number of reactive
355
sites exposed to the surface is low, leading to low Rβ-LG. Further, the osmotic environment
356
with the presence of LA and Ca2+ retain water molecules, preventing protein hydration. As a
357
result, in low RH, protein-protein interactions dominate, limiting the mobility of WPs. Ca2+
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ACCEPTED MANUSCRIPT on the other hand, forms salt bridges between carboxyl groups of amino acids (Zhu and
359
Damodaran, 1994), restricting the collision in required orientation for permanent covalent
360
bonding. Furthermore, anionic lactate actively increases the negative charge at the dimer
361
interface, stabilizing the β-LG dimers (Mercadante et al., 2012) and thereby, prevent the
362
formation of reactive monomers. Salty-WPC, is characteristically high in Na+ (approximately
363
0.5M), which exerts an osmotic pressure of ~0.005 bars, nearly 3 times greater compared to
364
that of sweet-WPC. Under these conditions, Na+ ions are preferentially excluded from the
365
protein surface which affects the binding of water to the protein (Arakawa and Timasheff,
366
1982). In addition, Na+ itself can interact with proteins through salt bridges and charge
367
shielding, subsequently inducing conformational changes and restricting the mobility of the
368
proteins. Influence of compositional variations of acid- and salty-WPCs on β-LG reaction
369
kinetics is further evident by the reaction order and Eo. Reaction orders of acid- and salty-
370
WPCs were approximately closer to 2 under all storage conditions, whereas, that in native-
371
and sweet-WPCs was approximately closer to 1. Furthermore, native-WPC required the
372
greatest Eo compared to other WPCs, implying that β-LG in native-WPC are still in their
373
native form, due to absence of severe thermal treatments during its manufacturing process.
374
Moreover, native-WPC consisted of 84% proteins on dry basis, implying the lack of
375
influence of other whey components on the β-LG structure. Compared to native- and sweet-
376
WPC, Eo was low in acid- and salty-WPCs. Compositional variations and heat treatments
377
applied in manufacturing processes of acid- and salty-WPCs partially denature the β-LG,
378
lowering the Eo.
379
In addition to β-LG, α-LA was also involved in protein aggregation in acid-WPC (Table 3),
380
with a higher rate of disappearance (Rα-LA) than β-LG. This observation contradicts the
381
assumption that in Ca2+ rich environment of acid-WPC α-LA should demonstrate the least
382
reactivity due to the high structural stability attained with the Ca2+ bound holo-form.
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ACCEPTED MANUSCRIPT However, low Eo established for α-LA in the current study suggests structural instabilities.
384
Perhaps folding intermediates of α-LA with a low affinity for Ca2+ (McSweeney, 2013) are
385
more common than the native form in acid-WPC. Rate of disappearance of α-LA (Rα-LA)
386
varied as 4 < 25 < 45 °C and 33 < 22 %. At high temperature, unfolding rate of α-LA
387
increased, shifting the equilibrium of α-N ⇌ α-U towards the right side (Oldfield et al.,
388
1998), in which state α-LA becomes more prone to engage in thiol/disulphide interchange
389
reactions with reactive β-LG.
390
4.0 Conclusion
391
Origin, composition and storage conditions clearly affected the properties of WPCs
392
examined. Protein aggregation occurred predominantly through covalent crosslinking under
393
all storage temperatures, while higher temperatures accelerated aggregation. Humidity had a
394
variable effect on the protein aggregation, mainly based on the type of WPC. Disulphide and
395
non-disulphide covalent crosslinking of β-LG was the main aggregation pathway in all
396
WPCs, whereas, involvement of α-LA, BSA and caseins depended on the type of WPC. In
397
parallel to the protein aggregation, disappearance of β-LG followed a first-order reaction in
398
native-WPC and sweet-WPC, while it was closely a second-order reaction in acid-WPC and
399
salty-WPC. Compositional variations associated with origin of acid-WPC and salty-WPC
400
facilitates the β-LG loss during the storage. According to the result of the current study, 4 °C
401
can be considered as the optimal storage temperature for all types of WPCs, while
402
determination of optimal relative humidity requires further understanding about effects of
403
storage on physical properties and functional characteristics of these powders.
404
5.0 Acknowledgement
405
The authors acknowledge the financial support granted by the Victoria University
406
Postgraduate Research Funding Scheme. Further gratitude extended to the Greek Yoghurt
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ACCEPTED MANUSCRIPT and Cheese Manufacturing Companies which contributed with providing whey stream
408
samples. Special acknowledgement is extended to the Dairy Innovation Australia Limited
409
(DIAL) for providing whey processing and spray drying facilities.
410
6.0 Reference
411
Arakawa, T., Timasheff, S.N., (1982). Preferential interactions of proteins with salts in
412
concentrated solutions. Biochemistry 21(25), 6545-6552.
413
Boye, J., Alli, I., (2000). Thermal denaturation of mixtures of α-lactalbumin and β-
414
lactoglobulin: a differential scanning calorimetric study. Food research international 33(8),
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673-682.
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Burgain, J., El Zein, R., Scher, J., Petit, J., Norwood, E.-A., Francius, G., Gaiani, C., (2016).
417
Local modifications of whey protein isolate powder surface during high temperature storage.
418
Journal of Food Engineering 178, 39-46.
419
Creamer, L.K., Bienvenue, A., Nilsson, H., Paulsson, M., van Wanroij, M., Lowe, E.K.,
420
Anema, S.G., Boland, M.J., Jiménez-Flores, R., (2004). Heat-induced redistribution of
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disulfide bonds in milk proteins. 1. Bovine β-lactoglobulin. Journal of Agricultural and Food
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Chemistry 52(25), 7660-7668.
423
Israelachvili, J.N., (2011). Intermolecular and surface forces: revised third edition. Academic
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press.
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Le, T.T., Deeth, H.C., Bhandari, B., Alewood, P.F., Holland, J.W., (2012). A proteomic
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approach to detect lactosylation and other chemical changes in stored milk protein
427
concentrate. Food Chemistry 132(1), 655-662.
428
Lefèvre, T., Subirade, M., (1999). Structural and interaction properties of β‐Lactoglobulin as
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studied by FTIR spectroscopy. International journal of food science & technology 34(5‐6),
430
419-428.
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ACCEPTED MANUSCRIPT Matsudomi, Oshita, T., Kobayashi, K., (1994). Synergistic interaction between β-
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lactoglobulin and bovine serum albumin in heat-induced gelation. Journal of Dairy Science
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77(6), 1487-1493.
434
Matsudomi, N., Oshita, T., Kobayashi, K., Kinsella, J.E., (1993). . alpha.-Lactalbumin
435
enhances the gelation properties of bovine serum albumin. Journal of Agricultural and Food
436
Chemistry 41(7), 1053-1057.
437
McSweeney, P.L.H., Fox P.F, (2013). Advanced Dairy Chemistry. Spinger, London.
438
Mercadante, D., Melton, L.D., Norris, G.E., Loo, T.S., Williams, M.A., Dobson, R.C.,
439
Jameson, G.B., (2012). Bovine β-lactoglobulin is dimeric under imitative physiological
440
conditions: dissociation equilibrium and rate constants over the pH range of 2.5–7.5.
441
Biophysical journal 103(2), 303-312.
442
Ngarize, S., Herman, H., Adams, A., Howell, N., (2004). Comparison of changes in the
443
secondary structure of unheated, heated, and high-pressure-treated β-lactoglobulin and
444
ovalbumin proteins using Fourier transform Raman spectroscopy and self-deconvolution.
445
Journal of Agricultural and Food Chemistry 52(21), 6470-6477.
446
Nishanthi, M., Chandrapala, J., Vasiljevic, T., (2017a). Compositional and structural
447
properties of whey proteins of sweet, acid and salty whey concentrates and their respective
448
spray dried powders. International Dairy Journal.
449
Nishanthi, M., Vasiljevic, T., Chandrapala, J., (2017b). Properties of whey proteins obtained
450
from different whey streams. International Dairy Journal 66, 76-83.
451
Norwood, E.-A., Chevallier, M., Le Floch-Fouéré, C., Schuck, P., Jeantet, R., Croguennec,
452
T., (2016). Heat-induced aggregation properties of whey proteins as affected by storage
453
conditions of whey protein isolate powders. Food and Bioprocess Technology 9(6), 993-
454
1001.
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ACCEPTED MANUSCRIPT Oldfield, D., Taylor, M., Singh, H., (2005). Effect of preheating and other process parameters
456
on whey protein reactions during skim milk powder manufacture. International Dairy Journal
457
15(5), 501-511.
458
Oldfield, D.J., Singh, H., Taylor, M.W., Pearce, K.N., (1998). Kinetics of denaturation and
459
aggregation of whey proteins in skim milk heated in an ultra-high temperature (UHT) pilot
460
plant. International Dairy Journal 8(4), 311-318.
461
Townend, R., Timasheff, S.N., (1960). Molecular Interactions in β-Lactoglobulin. III. Light
462
Scattering Investigation of the Stoichiometry of the Association between pH 3.7 and 5.22.
463
Journal of the American Chemical Society 82(12), 3168-3174.
464
Zhou, P., Liu, X., Labuza, T.P., (2008). Effects of moisture-induced whey protein
465
aggregation on protein conformation, the state of water molecules, and the microstructure and
466
texture of high-protein-containing matrix. Journal of Agricultural and Food Chemistry
467
56(12), 4534-4540.
468
Zhu, H., Damodaran, S., (1994). Effects of calcium and magnesium ions on aggregation of
469
whey protein isolate and its effect on foaming properties. Journal of Agricultural and Food
470
Chemistry 42(4), 856-862.
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ACCEPTED MANUSCRIPT Table 1: Composition of four WPCs Sweet-WPC
Acid-WPC
Salty-WPC
Moisture (%)
5.5 ± 0.7ab
4.1 ± 0.2ac
5.7 ± 0.8b
3.9 ± 0.08c
Total protein (%)
84.1 ± 0.6a
61.8 ± 0.1b
67 ± 0.3c
10.9 ± 0.2d
Lactose (%)
0.1 ± 0.02a
2.9 ± 0.03b
0.001 ± 0.0001a
0.1 ± 0.03b
Ca (%)
0.04 ± 0.001a
0.02 ± 0.003b
Na (%)
0.01 ± 0.004a
0.4 ± 0.02a
0.03 ± 0.01b
1.1 ± 0.05b
6.5 ± 0.03a
6.4 ± 0.02b
4.2 ± 0.02c
5.5 ± 0.01d
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Lactic acid (%)
pH
0.7 ± 0.02c
2.4 ± 0.3c
0.2 ± 0.02c
0.07 ± 0.001b
0.14 ± 0.01c
0.08 ± 0.001c
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Values are means of at least 4 independent measurements (n ≥ 4); the results are presented as means ± standard deviation (SD). Different lowercase superscripts in the same row depict the significant difference between means for each spray dried whey powder.
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Native-WPC
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ACCEPTED MANUSCRIPT Table 2: Reaction rate, reaction order, rate constant (Kn) activation energy (Eo) and pre- and
476
pre-exponential factor (K0) of β-LG disappearance in different WPCs during storage
33
22 Sweet 33
22
Order of reaction
Kn
Eo
Ink0
((g Kg-1)(1-n) day-1)
(kJ mol-1)
((g Kg-1)(1-n) day-1)
4
0.09e
1.4a
7.0 x 10-5 a
25
0.18f
1.0b
1.2 x 10-3 b
45
0.29g
0.7c
1.5 x 10-2 c
4
0.11h
1.3d
1.4 x 10-4 d
25
0.15i
1.2e
4.7 x 10-4 e
45
0.39j
0.6f
3.9 x 10-2 f
4
0.13a
1.2g
2.8 x 10-4 g
25
0.22n
45
0.24o
4
0.16p
25
0.19l
45
0.24q
4 25 45
Salty
4 33
25
22 Acid 33
SEM a-x
0.9h
3.2 x 10-3 h
0.8i
6.9 x 10-3 i
1.1j
5.4 x 10-4 j
0.9k
2.6 x 10-3 k
0.8l
5.3 x 10-3 l
0.14b
1.2m
5.8 x 10-4 m
0.16k
1.1n
6.8 x 10-4 n
0.20l
1.0o
2.2 x 10-3 o
0.07d
1.7p
3.3 x 10-5 p
0.08m
1.6q
5.9 x 10-5 q
0.11h
1.4r
2.1 x 10-4 r
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Native
Reaction rate for β-LG (µg / day)
96.3a
32.2a
97.6b
32.9b
58.3c
17.3c
41.2d
10.5d
23.1e
2.4e
31.9f
3.4f
13.2g
3.2g
54.5h
10.2h
0.1
0.04
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22
Temperature (°C)
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4
0.12a
1.4s
1.3 x 10-4 s
25
0.13a
1.4t
1.9 x 10-4 p
45
0.14b
1.3u
2.8 x 10-4 t
4
0.06c
1.9v
1.7 x 10-6 u
25
0.06d
1.9w
4.5 x 10-6 v
45
0.10e
1.6x
3.8 x 10-5 w
0.001
0.0
1.4 x 10-3
Different lowercase superscripts in the same column depicts the significant differences between means of kinetic parameters. Results are expressed as means of two independent replications. SEM = Standard error of the mean.
477
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Table 3: Reaction rate, reaction order, rate constant (Kn) activation energy (E0) and pre-
479
exponential factor (K0) of α-LA disappearance in acid-WPCs during storage Reaction rate for α-LA (µg / day)
Order of reaction
Kn
E0
Ink0
((g Kg-1)(1-n) day-1)
(kJ mol-1)
((g Kg-1)(1-n) day-1)
4
0.15a
1.6a
9.4 x 10-5 a
25
0.19b
1.5b
1.9 x 10-4 b
45
0.20c
1.5c
3.9 x 10-4 c
4
0.13d
1.7d
6.4 x 10-5 d
25
0.17e
1.7e
8.7 x 10-5 e
45
0.18f
1.6f
1.7 x 10-4 f
0.001
0.0
7.9 x 10-4
Acid 33
SEM a-f
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Temperature (0C)
25.3 ± 0
1.7 ± 0
17.4 ± 0.1
2.2 ± 0.04
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RH (%)
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Figure 1: Vector normalized, smoothened and deconvoluted FTIR spectra of the Amide I
485
region (1600 – 1700 cm-1) for NWP over storage period of 0 (solid- black), 14 (long dashed-
486
black), 30 (short dashed- black), 60 (solid- grey) and 90 day (dashed- grey) under different
487
storage temperatures (4, 25 and 45 0C) and RH (22 and 33 %)
488
Figure 2: Vector normalized, smoothened and deconvoluted FTIR spectra of the Amide I
489
region (1600 – 1700 cm-1) for SWP over storage period of 0 (solid- black), 14 (long dashed-
490
black), 30 (short dashed- black), 60 (solid- grey) and 90 day (dashed- grey) under different
491
storage temperatures (4, 25 and 45 0C) and RH (22 and 33 %)
492
Figure 3: Vector normalized, smoothened and deconvoluted FTIR spectra of the Amide I
493
region (1600 – 1700 cm-1) for AWP over storage period of 0 (solid- black), 14 (long dashed-
494
black), 30 (short dashed- black), 60 (solid- grey) and 90 day (dashed- grey) under different
495
storage temperatures (4, 25 and 45 0C) and RH (22 and 33 %)
496
Figure 4: Vector normalized, smoothened and deconvoluted FTIR spectra of the Amide I
497
region (1600 – 1700 cm-1) for StWP over storage period of 0 (solid- black), 14 (long dashed-
498
black), 30 (short dashed- black), 60 (solid- grey) and 90 day (dashed- grey) under different
499
storage temperatures (4, 25 and 45 0C) and RH (22 and 33 %)
500
Figure 5: SDS Non-reduced PAGE patterns of whey protein powders at 0, 30 and 90 days of
501
storage. L1, L2, L3, L4 represent NWP, SWP, AWP and StWP under 22% RH, while L5, L6,
502
L7, L8 represent NWP, SWP, AWP and StWP under 33% RH, respectively. X1 represent
503
covalently linked aggregates with Mw ≥250 kDa. X2 represents medium size protein
504
aggregates with Mw~35 – 67 kDa. X3 represent β-LG A.
505
Figure 6: SDS Reduced PAGE patterns of whey protein powders at 0, 30 and 90 days of
506
storage. L1, L2, L3, L4 represent NWP, SWP, AWP and StWP under 22% RH, while L5, L6,
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ACCEPTED MANUSCRIPT L7, L8 represent NWP, SWP, AWP and StWP under 33% RH, respectively. NDCA represent
508
non-disulphide covalently linked aggregates.
509
Figure 7: Changes in absolute quantity (µg) of non-reducible aggregates during storage of
510
NWP (solid line), SWP (dotted line), AWP (dashed line) and StWP (dashed-dotted line) at
511
storage temperatures of 4, 25 and 450C and relative humidity of 22 and 33%, as obtained
512
from image analysis of reducing SDS-PAGE.
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Figure 4:
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530 531 532
Figure 5.
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ACCEPTED MANUSCRIPT Highlights Properties of various WPCs were examined under different storage conditions.
•
β-LG was primarily involved in covalent cross linking in all WPCs.
•
Participation of α-LA in covalent crosslinking was evident primarily in acid-WPC
•
High storage temperature accelerated covalent linking between whey proteins
•
Reaction order of β-LG denaturation in acid and salty-WPCs was approximately 2
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S0260-8774(17)30278-9
DOI:
10.1016/j.jfoodeng.2017.06.032
Reference:
JFOE 8937
To appear in:
Journal of Food Engineering
Please cite this article as: Manjula Nishanthi, Jayani Chandrapala, Todor Vasiljevic, Properties of whey protein concentrate powders obtained by spray drying of sweet, salty and acid whey under varying storage conditions, Journal of Food Engineering (2017), doi: 10.1016/j.jfoodeng.2017.06.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Properties of whey protein concentrate powders obtained by spray drying of sweet,
2
salty and acid whey under varying storage conditions
3
Manjula Nishanthi1, Jayani Chandrapala1,2, Todor Vasiljevic1*
5
6
1
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University, Werribee campus, Victoria 3030, Australia
8
2
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Advanced Food Systems Research Unit, College of Health and Biomedicine, Victoria
M AN U
School of Science, RMIT University, Bundoora, Victoria 3083, Australia
9 10 11
*Corresponding Author:
Tel: +61 3 9919 8062
Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract
20
Changes of secondary structure and protein interactions of whey protein (WPs) present in
21
native, sweet, acid and salty-WPCs were analyzed following storage at 4, 25 or 45°C and 22
22
or 33% relative humidity for a period of 90-days. WPs aggregated predominantly through
23
covalent crosslinking, achieving maximum at 45 °C and 33% RH. Greater participation of β-
24
lactoglobulin in covalent crosslinking was evident in all WPCs, while that of α-lactalbumin
25
was significantly (p<0.05) high in acid-WPC only. Reaction order of β-LG denaturation in
26
acid and salty-WPCs was approximately 2 while approximately 1 in native and sweet-WPCs.
27
Activation energy was significantly (p<0.05) higher in native and sweet-WPCs, with
28
averages recorded as 97 and 49.8 kJ mol-1, respectively, than that in acid and salty-WPCs with
29
averages of 27.5 and 33.8 kJ mol-1, respectively, mainly attributed to inherently high
30
concentrations of lactic acid and salts in these WPCs.
31
Key word: Whey protein concentrate, protein aggregation, covalent interactions, acid whey,
32
salty whey
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ACCEPTED MANUSCRIPT Introduction:
35
Whey protein concentrates (WPCs) are an important protein source used in many food
36
systems due to their excellent functional characteristics such as emulsification, gelling and
37
foaming. Dairy industry has been successful in utilizing the sweet whey waste stream for
38
manufacturing of various WPCs. However, the industry has not been very successful in
39
utilizing acid and salty whey in an equivalent way to sweet whey, mainly due to their
40
composition-induced processing obstacles such as high acidity and salinity, respectively.
41
Moreover, the powders produced from these two streams form clumps and sticky deposits
42
during storage. A recent study by Nishanthi et al. (2017a) showed that the particle surface of
43
salty-WPC was hydrophobic mainly due to high surface fat content, while that of acid-WPC
44
was characterized by both hydrophobic and hydrophilic nature due to presence of elevated
45
protein concentration. Therefore, these two WPCs possess a high potential to be used as
46
functional ingredients in food systems. Thus, a thorough fundamental understanding on the
47
stability of proteins present in these respective WPCs as affected by storage is a necessity in
48
evaluating the potential of using these two particular WPCs.
49
Storage conditions induce changes in protein conformation at a molecular level, which in turn
50
may affect functional properties of the protein. Intermolecular covalent and non-covalent
51
interactions occur between whey proteins (WPs), resulting in protein unfolding and
52
denaturation, eventually leading to reversible and irreversible aggregation. As a result, a
53
proportion of native WPs decreases after 3 months of storage at 40 °C with a concomitant
54
increase in proportion of denatured proteins. These changes were clearly temperature
55
dependent as aforementioned changes were not noticed at 4 °C or 20 °C up to 15 months of
56
storage (Norwood et al., 2016). At elevated storage temperatures, flexibility and mobility of
57
WPs increases, resulting in partial loss of their tertiary structure (Norwood et al., 2016). WPC
58
was found to have increased surface hydrophobicity and less exposed amino acids on the
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ACCEPTED MANUSCRIPT particle surfaces after storage at 60 °C and 22 % RH for one month (Burgain et al., 2016). In
60
addition to denaturation, WPs can be subjected to lactosylation during storage. While studies
61
on lactosylation of WPC powders are lacking, Le et al. (2012) reported that six out of twelve
62
lysine residues of α-lactalbumin (α-LA) would lactosylate during storage of milk protein
63
concentrates up to 12 weeks at 40 °C and 84 % RH, which indicates that similar changes
64
could be expected in WPC, especially those containing greater lactose content. These
65
structural modifications in WPs could subsequently affect their functional properties. For
66
instance, formation of insoluble aggregates in response to elevated storage temperatures
67
reduced the solubility of WPC (Zhou et al., 2008) affecting several functional characteristics
68
such as emulsification, gelation and foaming.
69
So far, the studies mainly focused on WPC produced from the sweet whey and no study thus
70
far has established physico-chemical properties of WPs during storage of WPCs produced
71
from acid or salty whey, which were identified as potentially effective functional ingredients
72
(Nishanthi et al., 2017a). Therefore, the main aim of the current study was to establish
73
conformational and surface interactional changes of WPs present in sweet, acid and salty
74
WPCs under three different storage temperatures (4, 25, 45 °C) and two relative humidities
75
(RH) (22% and 33%) over a storage period of 90 days. Native-WPC was used as the
76
reference sample.
77
2. Methodology
78
2.1 Sample preparation
79
Four WPCs, native-WPC, sweet-WPC, acid-WPC and salty-WPC were manufactured in our
80
previous study (Nishanthi et al., 2017a) with the composition shown in Table 1 and used
81
during storage trial in the current study. WPCs were first completely dehydrated by placing
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ACCEPTED MANUSCRIPT appropriate quantity of samples in vacuum desiccators over phosphorous pentoxide at room
83
temperature (25°C) for 4 weeks.
84
2.2 Storage trial
85
Approximately 3 g of each WPC was placed in standard desiccators (with a porcelain disc
86
size of 250 mm) with a controlled RH (22 and 33%), sealed and placed in temperature
87
controlled cabinets at three temperatures (4, 25 and 45°C). The equilibrium RH in each
88
desiccator was maintained using saturated salt slurries containing potassium acetate (22 ±
89
2%) or magnesium chloride (33 ± 1%). At 4, 25 and 45 °C, magnesium chloride maintained
90
RH of 34, 33 and 32%, while potassium acetate maintained RH of 24, 23 and 23%,
91
respectively. Five desiccators were prepared for each temperature/humidity combination as
92
sampling was conducted at 5 time points representing 0, 14, 30, 60 and 90 days of storage. A
93
pre-trial test was conducted to estimate the equilibrium time and identify the first sampling
94
point. Several samples from each WPC were stored in standard desiccators with saturated salt
95
slurries of magnesium chloride and potassium acetate. After one week of storage, one sample
96
from each WPC was tested for the water activity using a water activity meter (Aqualab CX-
97
3T, Labcell, Hampshire, United Kingdom) at 25°C each day. The four types of WPCs reach
98
to the water activity of 0.22 and 0.33 by one week and four days. A stabilised water activity
99
level could be achieved within two weeks. Therefore, the first sampling (0 day) was
100
conducted after allowing 2-weeks equilibrium period from the initiation date of the storage
101
trial. After opening desiccators at a required time point, the analysis was carried out within 3
102
days.
103
2.3 Fourier Transform Infrared Spectroscopy (FTIR)
104
The FTIR spectra of WPCs were obtained in the range of 4000 - 600 cm−1 using the
105
PerkinElmer Frontier FTIR spectrometer (PerkinElmer, MA, USA) as described previously
106
(Nishanthi et al., 2017b). The spectra were vector normalized, smoothened and deconvoluted
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ACCEPTED MANUSCRIPT with the aid of IR Solution software (Shimadzu Corporation, Kyoto, Japan), in order to
108
recognize the corresponding peaks within the amide I region between 1600 and 1700 cm−1.
109
2.4 Sodium Dodecyl Sulphate Gel Electrophoresis (SDS – PAGE)
110
In order to establish non-covalent and covalent interactions, Sodium Dodecyl Sulphate (SDS)
111
polyacrylamide gel electrophoresis (PAGE) was performed under reducing (SDSR) and non-
112
reducing (SDSNR) conditions using freshly casted gels with 30% (w/v) acrylamide and 10%
113
(w/v) SDS (Nishanthi et al., 2017b). Samples were reduced with addition of β-
114
mercaptoethanol and subsequent heating at 100 °C for 5 min.
115
2.5 Kinetics analysis
116
The rate (1) and the Arrhenius equations (2) were used to calculate the kinetic parameters
117
involved in disappearance of native WPs during storage of WPCs (Oldfield et al., 2005).
118
− = (1)
119
Where:
120
C = Protein concentration (g kg-1)
121
t = time (days)
122
kn = rate constant ((g kg-1)(1-n) day-1)
123
n = reaction order
124
125
= (2)
126
Where:
127
k0 = pre-exponential term ((g kg-1)(1-n) day-1)
128
Ea = activation energy (kJ mol-1)
129
R = universal Gas Constant (8.314 J mol-1 K-1)
130
T = temperature (K)
131
2.6 Statistical analysis
132
The experiments were organized in a randomized split plot blocked design with 3 main
133
factors: a type of WPC as the main plot, and temperature and RH as subplots. The
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135
of the kinetic and compositional analysis were statistically analyzed using the General Linear
136
Model (GLM) and ANOVA procedure, respectively. The level of significance was preset at
137
p<0.05.
138
3. Results and discussion:
139
3.1 Secondary structural changes during storage through FTIR
140
Significant and complex intermolecular interactions occur among WPs in response to storage
141
conditions and associated compositional changes of examined WPCs. These changes affect
142
the conformation of secondary structure motifs such as α-helices, β-sheets, β-turns and
143
random coils in these proteins as shown in Figures 1 to 4.
144
At the beginning of the storage trial, WPs present in native-WPC were characterized as β-
145
sheet rich proteins due to two prominent peaks observed in the regions of 1628-1629 cm-1
146
and 1693 cm-1 (Figure 1). However, other secondary structures, such as α-helixes, random
147
peptides and β-turn were largely missing at 4 °C, but appeared with low intensities at 25 and
148
45 °C, irrespective of RH. In addition to the native moieties, β-sheet cross linking (1682 cm-1
149
and 1615 cm-1) observed at the beginning of storage can be attributed to the thermal impacts
150
associated with pasteurization and spray drying applied during processing of this WPC.
151
During initial stages of the storage (14-30 days), secondary conformations did not change
152
substantially. However, after 60 days of storage WPs underwent several major structural
153
changes. These changes were more pronounced at 33% RH as compared to that at 22% RH,
154
but were independent of storage temperature. Firstly, complete distortion of α-helixes
155
occurred under all storage conditions, which in turn increased the intensity of randomly
156
arranged peptides (1644 cm-1). In presence of high amount of moisture (33 % RH),
157
hydrophilic amino acid residues of α-helix form hydrogen bonds with water molecules, thus
158
disturbing its intermolecular interactions. As a result of α-helical stretching, intensity of β-
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ACCEPTED MANUSCRIPT turns (1660 cm-1) increased attributed to the broadening of β-turn between β-strand I and H in
160
β-lactoglobulin (β-LG). Secondly, widening of peaks attributed to β-sheets, observed
161
prominently at 33% RH, suggests a possible intermolecular cross linking in the presence of
162
elevated moisture content. Concomitantly, intensity of anti-parallel β-sheets (Lefèvre and
163
Subirade, 1999) (1691 cm-1) increased, suggesting a dimerization or polymerization, moving
164
high number of β-sheets to the molecular interior. Thirdly, greater exposure of amino acid
165
(AA) side chain residues (1610 cm-1) (Lefèvre and Subirade, 1999) were observed mainly at
166
33% RH. The random coils formed by stretching α-helixes may be small with high exposed
167
surface area, which subsequently expose a high number of AA residues. Moreover, protein
168
hydration due to greater relative humidity leads to movement of hydrophilic AA residues to
169
the molecular exterior. At the end of the storage (90 days), the peaks representing β-turns and
170
random coils widened and disappeared suggesting a possible aggregation that destructed
171
native monomeric and dimeric forms of proteins. This loss of secondary structures was
172
especially magnified in the samples stored at 45 °C and 33% RH. Further, the exposed side
173
chain residues disappeared under all storage conditions. Interestingly, a greater content of β-
174
sheets remained unaffected, suggesting the preservation of the calyx of β-LG.
175
Sweet-WPC mainly contained intramolecular β-sheets (1630 and 1693 cm-1) and
176
intermolecular β-sheets (1615 cm-1) at the start of the storage (Figure 2). Interlinking of β-
177
sheets observed at the beginning of the storage suggests an impact of thermal treatments
178
associated with the manufacturing process of sweet-WPC (Nishanthi et al., 2017a). Similar to
179
native-WPC, initial storage period (14-30 days) did not change the profile of the secondary
180
structure of sweet-WPC to a great extent for most storage conditions, although random coils,
181
α-helixes and β-turns appeared in the samples stored at 33% RH. These changes can be
182
attributed to the protein hydration, leading acquisition of some native attributes. Furthermore,
183
a greater exposure of side chain AA residues was observed (1611 cm-1) at 22% RH. Similar
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ACCEPTED MANUSCRIPT to native-WPC, substantial structural changes were noticed after 60 days of storage.
185
Irrespective of storage conditions, β-sheets lost their conformational stability, partially
186
forming cross linked β-sheets (1680 cm-1). Strong modifications were seen in β-sheets
187
indicated by appearance of two peaks (1623 and 1634 cm-1), which can be attributed to the
188
disruption of intramolecular hydrogen bonds, leading to formation of new stronger
189
intermolecular hydrogen bonds (Ngarize et al., 2004). This modification was especially
190
prominent at 25 and 45 °C. Monomer-dimer equilibrium of β-LG molecules (Lefèvre and
191
Subirade, 1999) has shifted towards the dimeric form, resulting in strongly bonded β-strand
192
structures. Dimer formation, in parallel, leads to a high number of deeply buried β-type
193
structures (1691 cm-1). Moreover, dimer formation takes place at expense of intramolecular
194
α-helix, subsequently, resulting in high number of random coils. The α-helix stretching into
195
random coils was high at 25 and 45 °C, while independent of RH. Therefore, the dimerization
196
which was established in SWP after 60 days was a result of storage temperature. By the end
197
of storage, secondary structures other than β-sheets completely disappeared in the samples
198
stored at 25 and 45 °C. Furthermore, aging of sweet-WPC after 60 days induced a
199
conformational change to the calyx of β-LG indicated by a shift in peak at 1691 cm-1 to a
200
peak with low intensity at 1694 cm-1. This change in turn may lead to the exposure of reactive
201
sites for covalent and hydrophobic interactions.
202
In acid-WPC, β-sheets (1620 – 1635 cm-1) were largely absent at the beginning of the storage
203
(Figure 3). Simultaneously, aggregation between anti-parallel β-sheets (1682 cm-1) was
204
prominent at 22 % RH and 45 °C, involving mainly β-strands located on the surface, as
205
opposed to those in deep areas of β-LG (1691 cm-1), which remained intact. Other secondary
206
structures, such as α-helixes, β-turns and random coils were present at a low intensity,
207
regardless of the storage conditions. By 14 days of storage, additional exposure of β-sheets
208
(1621 cm-1) occurred suggesting a partial unfolding of β-LG in response to lactic acid (LA)-
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ACCEPTED MANUSCRIPT induced WP alterations (Nishanthi et al., 2017b). Unfolding of β-sheets was more prominent
210
at 33% RH as compared to that at 22% RH. Deprotonation of LA occurs rapidly in the
211
presence of moisture due to its low acid dissociation constant (pKa), which in turn decreases
212
the negative surface charge density of the proteins promoting attractive forces, inducing
213
protein aggregation driven by van der Waals and electrostatic interactions (Israelachvili,
214
2011). After 60 days of storage, three significant modifications were noticed. Firstly, α-
215
helixes disappeared completely forming non-native random structures under all storage
216
conditions. Secondly, conformation of β-sheets changed substantially, converting into
217
distinctive two peaks in the region of 1620 – 1638 cm-1, implying a dimerization of β-LG. In
218
addition, under all storage conditions the intensity of deeply located β-sheets (1693 cm-1) was
219
doubled. In presence of LA, β-LG formed dimers, relocating more of β-sheets to the inner
220
side of the molecule. Thirdly, side chain amino acid residues (1610 cm-1) were greatly
221
exposed, especially at 22% RH as a result of β-sheet modifications and α-helix disruption.
222
Towards the end of the storage (90 day), a greater loss of secondary structures was observed,
223
especially in the samples stored at 25 and 45 °C under 22% RH. A greater loss of random
224
coils and dimeric forms of β-LG suggested a severe aggregation.
225
In contrast to other WPCs, secondary structures of salty-WPC (Figure 4) showed high
226
sensitivity to storage conditions during the first days of storage. For instance, proportions of
227
β-sheets (1626, 1636 and 1693 cm-1), random peptides (1648 cm-1) and β-turns (1660 cm-1)
228
clearly declined as temperature and humidity increased. In parallel, a number of deeply
229
located β-sheets (1693 cm-1) descended with increase in temperature, denoting an exposure of
230
the β-LG core. However, the temperature-dependent reactivity of β-LG did not increase the
231
proportion of cross linked β-sheets (1616 and 1680 cm-1). Due to osmotic pressure caused by
232
Na+ in the medium, β-sheets did not interlink, instead unfolded, forming unordered structures,
233
as apparent by intensity of high random coil. A dimeric form of β-LG was also observed at 4
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ACCEPTED MANUSCRIPT and 25 °C (1620 – 1636 cm-1), as opposed to monomeric β-LG at 45 °C. Other secondary
235
motifs were also greatly absent at 45 °C. During further storage (14-30 days), monomeric β-
236
LG dominated (Lefèvre and Subirade, 1999) under all storage conditions, suggesting that
237
native-dimers dissociated with aging. With increase in temperature, concomitant reduction of
238
non-native monomers and enhanced aggregation driven by crosslinking of β-sheets (1616 cm-
239
1
240
storage, β-sheets reappeared in the region of 1620 – 1638 cm-1, whereas the peak intensity
241
increased with the storage temperature. Interestingly, the region denoted β-sheets (1620 –
242
1636 cm-1) consisted of two peaks at 25 and 45 °C, denoting dimeric forms of β-LG, while it
243
was just a single peak at 4 °C, attributed to monomeric β-LG. Dimeric β-LG formation in
244
turn increased the number of intermolecular β-sheets (1680 cm-1), resulting in substantially
245
greater proportion of deeply located β-sheets (1691 cm-1) and high number of random coils
246
and β-turns. Up to 60 days, the observed structural changes were independent of the
247
humidity. However, towards the end of storage (90 day), humidity influenced the interactions
248
among β-sheets. High occurrence of intermolecular hydrogen bonds prominent at 22% RH
249
suggested a high occurrence of β-LG dimers (1620 – 1636 cm-1), while intramolecular
250
hydrogen bonds predominated at 33 % RH, suggesting more of monomeric form of β-LG.
251
The prolonged exposure to high humidity reduced the protein-protein interactions, instead
252
allowing protein hydration and formation of near-native protein monomers. This assumption
253
is also supported by the occurrence of less cross linked β-sheets (1615 cm-1) at 33% RH.
254
3.2 Changes in protein interactions and aggregation during storage
255
At the beginning of the storage trial, aggregates (Mw≥250 kDa) covalently linked via
256
disulphide bridges were observed in native-WPC (Figure 5 and 6) accompanied with
257
subsequent disappearance of β-LG directly related to temperature, humidity and storage time.
258
Intensity of α-LA bands on the other hand remained approximately consistent during initial
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ACCEPTED MANUSCRIPT 30 days of storage, but started to decline after this period implying that α-LA would be
260
involved in the overall aggregation process at a later stage. It is known that α-LA contains
261
only four disulphide bonds positioned at Cys6-Cys120, Cys28-Cys11, Cys61-Cys77 and Cys73-
262
Cys91 (McSweeney, 2013), but no free thiol groups. In addition, the holo-form of α-LA,
263
which is present abundantly in native-WPC, provides a high thermal stability (Boye and Alli,
264
2000). For these reasons, involvement of α-LA in overall aggregation is limited. Small
265
amount of caseins was present in this WPC, which appeared to be involved in disulphide
266
linking over the storage period, with the greatest extent at 45 °C and 33% RH.
267
Thiol/disulphide exchange reaction usually occurs between Cys160 of β-LG and free thiols of
268
Cys11 and Cys88 of к-casein (Creamer et al., 2004) forming an aggregate with a minimum
269
Mw of ~ 37 kDa (Figure 5). Similar to other proteins, bovine serum albumin (BSA) was also
270
involved in aggregation. However, its involvement was clearly storage time dependent, while
271
other main factors had no effect. BSA forms disulphide crosslinks with β-LG (Matsudomi et
272
al., 1994) due to a free thiol group at Cys34. In addition to disulphide bridging, WPs in native-
273
WPC participated in non-reducible aggregation (Figure 7), likely due to lactosylation of
274
lysine residues in these proteins. Compared to other WPCs, lactosylation was less prominent
275
in this WPC without a clear association with storage temperature and time, which could have
276
been caused by a very low amount of lactose.
277
Sweet-WPC samples exhibited a similar behavior to that of native-WPC presented by clear
278
formation of protein aggregates covalently linked by disulphide bridging over the overall
279
storage period (Figure 5 and 6), again achieving maximum aggregation at 45 °C and 33%
280
RH. In parallel to the aggregate formation, a rate of β-LG disappearance was the greatest
281
under the same conditions (Table 2), suggesting that β-LG was the main protein involved in
282
aggregation. Conversely, only minor involvement of α-LA in aggregation was observed
283
under all storage conditions. No involvement of BSA was evident in sweet-WPC under all
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ACCEPTED MANUSCRIPT storage conditions, although comparatively a higher concentration of BSA was present in
285
comparison with other WPCs. In contrast, caseins were involved in disulphide crosslinking,
286
but a clear association with storage conditions and time could not be established. In addition
287
to aggregates linked by disulphide bonds, two distinct bands representing non-disulphide
288
linked aggregates appeared with approximate Mw of 71-72 kDa and ≥250 kDa (Figure 6).
289
Compared to native-WPC, these aggregates were prominent in sweet-WPC, showing a
290
gradual increase with rise in the storage temperature, humidity and time. Sweet-WPC
291
contained 2.9% of lactose, which might have led to lactosylation of lysine segments of β-LG.
292
Among these, Lys8 located at N terminal, Lys141 located at C terminal end and Lys138 located
293
in 3 turn α-helix are easily accessible, therefore, likely the primary sites of the reaction.
294
In acid-WPC, disulphide linked aggregates gradually increased throughout the storage with
295
subsequent disappearance of β-LG A, β-LG B and α-LA (Figure 5 and 6). Rapid involvement
296
of β-LG A was observed in aggregation, compared to β-LG B (Oldfield et al., 1998), mainly
297
due to disulphide bridging via Val118 in β-LA A as compared to Ala118 in β-LG B, located in
298
β-strand H, creating a less favorably packed core (McSweeney, 2013). This in turn increases
299
the flexibility of β-LG A and exposes the free thiol group. Compared to other WPCs,
300
involvement of α-LA in protein aggregation was greater in acid-WPC. Less amount of water
301
might have restricted the availability of Ca2+ to the binding site of α-LA, preventing protein
302
stabilization and thus resulting in prevalence of less stable apo-α-LA. The contribution of
303
caseins in disulphide bonding was apparent only at 45 °C and 33% RH conditions, although a
304
significantly higher amount of caseins was present in acid-WPC than in other WPCs
305
(Nishanthi et al., 2017a). A diffuse region was observed in the region between caseins and
306
BSA (Mw~50 kDa) in the samples stored at 45 °C and two RHs under reducing PAGE
307
conditions (Figure 6), suggesting a possible aggregation between caseins and β-LG and/or α-
308
LA via non-reducible covalent bonds. In addition to reducible aggregates, non-reducible
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ACCEPTED MANUSCRIPT aggregates likely created by lactosylation with Mw>250 kDa were observed in acid-WPC
310
under all storage condition (Figure 6). The extent of aggregation was clearly temperature and
311
RH dependent (Figure 7) and predominantly involved α-LA, as evidenced by the reaction
312
rates (Table 3). Out of twelve lysine residues in α-LA, maximum of 6 residues (Lys24, Lys112,
313
Lys117, Lys127, Lys133 and Lys141) are lactosylated easily (Le et al., 2012).
314
Similar to other WPCs, salty-WPC contained disulphide linked protein aggregates with
315
Mw>250 kDa (Figure 5 and 6). The aggregation increased with the storage time, achieving
316
the maximum at 45 °C and 33% RH. Both β-LG A and β-LG B participated in disulphide
317
bonding, however, β-LG A completely disappeared after 14 days, suggesting a high reaction
318
rate (Oldfield et al., 1998). Involvement of α-LA in disulphide bonding was observed with
319
extent clearly dependent of the storage temperature and time with no clear association with
320
humidity. Thus, thiol/disulphide interchange reactions between β-LG and α-LA could be the
321
main possible aggregation pathway. In absence of a reactive thiol group, thiol/disulphide
322
interchange reactions occur through the disulphide bonds of Cys6-Cys120 and Cys28-Cys111
323
located on the surface of α-LA, which form new disulphide bonds with Cys66 and Cys160
324
located on the surface of β-LG. Compared to other WPCs, salty-WPC contained a high
325
concentration of lactoferrin (LF) (Mw~87 kDa), which did not participate in covalent
326
aggregation under all storage conditions. On the other hand, BSA was greatly involved
327
concomitantly with the rise in storage temperature, humidity and time. Due to a free thiol
328
group at Cys34 and seventeen disulphide bonds in its primary structure, BSA participates in
329
both disulphide bridging and thiol/disulphide interchange reactions with β-LG (Matsudomi et
330
al., 1994) and α-LA (Matsudomi et al., 1993). Similar to sweet-WPC, salty-WPC contained
331
aggregates linked via covalent non-disulphide bonds in the region of BSA-LF with
332
approximate Mw of 71-72 kDa. Furthermore, the amount of large aggregates (Mw≥250 kDa)
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ACCEPTED MANUSCRIPT formed via lactosylation increased in parallel with the storage temperature, humidity and
334
time.
335
In four WPCs, type of proteins and interactions involved in protein aggregation vary with the
336
type of powder, storage conditions and time. The main protein involved in disulphide and
337
non-disulphide covalent aggregation was β-LG. Therefore, kinetic parameters of β-LG were
338
estimated to thoroughly understand the differences in behavior of β-LG in these WPCs.
339
3.3. Kinetics of β-LG disappearance
340
Kinetic parameters of β-LG disappearance in different WPCs during storage are presented in
341
Table 2. Disappearance of β-LG follows the common reaction kinetic principles. A
342
substantial reaction occurs if an adequate number of β-LG molecules with sufficient
343
activation energy (Eo) collide at an adequate frequency while maintaining specified molecular
344
orientation to form disulphide or other covalent bonds. Rate of disappearance of β-LG (Rβ-LG)
345
in native-WPC and sweet-WPC was directly related to the storage temperature and humidity,
346
being the greatest at 45 °C and 33% RH. Elevated temperature increases the number of
347
reactive β-LG molecules with exposed free thiol group of Cys121. High RH increases water
348
activity thus favors thermal energy transition and molecular motions, facilitating formation of
349
reactive monomers. In contrast, Rβ-LG of salty-WPC and acid-WPC decreases as RH
350
increases. This observation suggests a greater interference of compositional constituents
351
present in these two WPCs on Rβ-LG. As a result of the low pH in presence of high
352
concentration of LA in acid-WPC (Nishanthi et al., 2017a), dimerization of β-LG and
353
formation of octamers (Townend and Timasheff, 1960) occurs as evidenced by the FTIR
354
spectra and SDSNR PAGE patterns. Due to the large structure of octamer, number of reactive
355
sites exposed to the surface is low, leading to low Rβ-LG. Further, the osmotic environment
356
with the presence of LA and Ca2+ retain water molecules, preventing protein hydration. As a
357
result, in low RH, protein-protein interactions dominate, limiting the mobility of WPs. Ca2+
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ACCEPTED MANUSCRIPT on the other hand, forms salt bridges between carboxyl groups of amino acids (Zhu and
359
Damodaran, 1994), restricting the collision in required orientation for permanent covalent
360
bonding. Furthermore, anionic lactate actively increases the negative charge at the dimer
361
interface, stabilizing the β-LG dimers (Mercadante et al., 2012) and thereby, prevent the
362
formation of reactive monomers. Salty-WPC, is characteristically high in Na+ (approximately
363
0.5M), which exerts an osmotic pressure of ~0.005 bars, nearly 3 times greater compared to
364
that of sweet-WPC. Under these conditions, Na+ ions are preferentially excluded from the
365
protein surface which affects the binding of water to the protein (Arakawa and Timasheff,
366
1982). In addition, Na+ itself can interact with proteins through salt bridges and charge
367
shielding, subsequently inducing conformational changes and restricting the mobility of the
368
proteins. Influence of compositional variations of acid- and salty-WPCs on β-LG reaction
369
kinetics is further evident by the reaction order and Eo. Reaction orders of acid- and salty-
370
WPCs were approximately closer to 2 under all storage conditions, whereas, that in native-
371
and sweet-WPCs was approximately closer to 1. Furthermore, native-WPC required the
372
greatest Eo compared to other WPCs, implying that β-LG in native-WPC are still in their
373
native form, due to absence of severe thermal treatments during its manufacturing process.
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Moreover, native-WPC consisted of 84% proteins on dry basis, implying the lack of
375
influence of other whey components on the β-LG structure. Compared to native- and sweet-
376
WPC, Eo was low in acid- and salty-WPCs. Compositional variations and heat treatments
377
applied in manufacturing processes of acid- and salty-WPCs partially denature the β-LG,
378
lowering the Eo.
379
In addition to β-LG, α-LA was also involved in protein aggregation in acid-WPC (Table 3),
380
with a higher rate of disappearance (Rα-LA) than β-LG. This observation contradicts the
381
assumption that in Ca2+ rich environment of acid-WPC α-LA should demonstrate the least
382
reactivity due to the high structural stability attained with the Ca2+ bound holo-form.
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ACCEPTED MANUSCRIPT However, low Eo established for α-LA in the current study suggests structural instabilities.
384
Perhaps folding intermediates of α-LA with a low affinity for Ca2+ (McSweeney, 2013) are
385
more common than the native form in acid-WPC. Rate of disappearance of α-LA (Rα-LA)
386
varied as 4 < 25 < 45 °C and 33 < 22 %. At high temperature, unfolding rate of α-LA
387
increased, shifting the equilibrium of α-N ⇌ α-U towards the right side (Oldfield et al.,
388
1998), in which state α-LA becomes more prone to engage in thiol/disulphide interchange
389
reactions with reactive β-LG.
390
4.0 Conclusion
391
Origin, composition and storage conditions clearly affected the properties of WPCs
392
examined. Protein aggregation occurred predominantly through covalent crosslinking under
393
all storage temperatures, while higher temperatures accelerated aggregation. Humidity had a
394
variable effect on the protein aggregation, mainly based on the type of WPC. Disulphide and
395
non-disulphide covalent crosslinking of β-LG was the main aggregation pathway in all
396
WPCs, whereas, involvement of α-LA, BSA and caseins depended on the type of WPC. In
397
parallel to the protein aggregation, disappearance of β-LG followed a first-order reaction in
398
native-WPC and sweet-WPC, while it was closely a second-order reaction in acid-WPC and
399
salty-WPC. Compositional variations associated with origin of acid-WPC and salty-WPC
400
facilitates the β-LG loss during the storage. According to the result of the current study, 4 °C
401
can be considered as the optimal storage temperature for all types of WPCs, while
402
determination of optimal relative humidity requires further understanding about effects of
403
storage on physical properties and functional characteristics of these powders.
404
5.0 Acknowledgement
405
The authors acknowledge the financial support granted by the Victoria University
406
Postgraduate Research Funding Scheme. Further gratitude extended to the Greek Yoghurt
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ACCEPTED MANUSCRIPT and Cheese Manufacturing Companies which contributed with providing whey stream
408
samples. Special acknowledgement is extended to the Dairy Innovation Australia Limited
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(DIAL) for providing whey processing and spray drying facilities.
410
6.0 Reference
411
Arakawa, T., Timasheff, S.N., (1982). Preferential interactions of proteins with salts in
412
concentrated solutions. Biochemistry 21(25), 6545-6552.
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Boye, J., Alli, I., (2000). Thermal denaturation of mixtures of α-lactalbumin and β-
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lactoglobulin: a differential scanning calorimetric study. Food research international 33(8),
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673-682.
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Burgain, J., El Zein, R., Scher, J., Petit, J., Norwood, E.-A., Francius, G., Gaiani, C., (2016).
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Local modifications of whey protein isolate powder surface during high temperature storage.
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Journal of Food Engineering 178, 39-46.
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Creamer, L.K., Bienvenue, A., Nilsson, H., Paulsson, M., van Wanroij, M., Lowe, E.K.,
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Anema, S.G., Boland, M.J., Jiménez-Flores, R., (2004). Heat-induced redistribution of
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disulfide bonds in milk proteins. 1. Bovine β-lactoglobulin. Journal of Agricultural and Food
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Chemistry 52(25), 7660-7668.
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Israelachvili, J.N., (2011). Intermolecular and surface forces: revised third edition. Academic
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Le, T.T., Deeth, H.C., Bhandari, B., Alewood, P.F., Holland, J.W., (2012). A proteomic
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approach to detect lactosylation and other chemical changes in stored milk protein
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concentrate. Food Chemistry 132(1), 655-662.
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Lefèvre, T., Subirade, M., (1999). Structural and interaction properties of β‐Lactoglobulin as
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studied by FTIR spectroscopy. International journal of food science & technology 34(5‐6),
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419-428.
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ACCEPTED MANUSCRIPT Matsudomi, Oshita, T., Kobayashi, K., (1994). Synergistic interaction between β-
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lactoglobulin and bovine serum albumin in heat-induced gelation. Journal of Dairy Science
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77(6), 1487-1493.
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Matsudomi, N., Oshita, T., Kobayashi, K., Kinsella, J.E., (1993). . alpha.-Lactalbumin
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enhances the gelation properties of bovine serum albumin. Journal of Agricultural and Food
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Chemistry 41(7), 1053-1057.
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McSweeney, P.L.H., Fox P.F, (2013). Advanced Dairy Chemistry. Spinger, London.
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Mercadante, D., Melton, L.D., Norris, G.E., Loo, T.S., Williams, M.A., Dobson, R.C.,
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Jameson, G.B., (2012). Bovine β-lactoglobulin is dimeric under imitative physiological
440
conditions: dissociation equilibrium and rate constants over the pH range of 2.5–7.5.
441
Biophysical journal 103(2), 303-312.
442
Ngarize, S., Herman, H., Adams, A., Howell, N., (2004). Comparison of changes in the
443
secondary structure of unheated, heated, and high-pressure-treated β-lactoglobulin and
444
ovalbumin proteins using Fourier transform Raman spectroscopy and self-deconvolution.
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Journal of Agricultural and Food Chemistry 52(21), 6470-6477.
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Nishanthi, M., Chandrapala, J., Vasiljevic, T., (2017a). Compositional and structural
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properties of whey proteins of sweet, acid and salty whey concentrates and their respective
448
spray dried powders. International Dairy Journal.
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Nishanthi, M., Vasiljevic, T., Chandrapala, J., (2017b). Properties of whey proteins obtained
450
from different whey streams. International Dairy Journal 66, 76-83.
451
Norwood, E.-A., Chevallier, M., Le Floch-Fouéré, C., Schuck, P., Jeantet, R., Croguennec,
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T., (2016). Heat-induced aggregation properties of whey proteins as affected by storage
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conditions of whey protein isolate powders. Food and Bioprocess Technology 9(6), 993-
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1001.
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ACCEPTED MANUSCRIPT Oldfield, D., Taylor, M., Singh, H., (2005). Effect of preheating and other process parameters
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on whey protein reactions during skim milk powder manufacture. International Dairy Journal
457
15(5), 501-511.
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Oldfield, D.J., Singh, H., Taylor, M.W., Pearce, K.N., (1998). Kinetics of denaturation and
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aggregation of whey proteins in skim milk heated in an ultra-high temperature (UHT) pilot
460
plant. International Dairy Journal 8(4), 311-318.
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Townend, R., Timasheff, S.N., (1960). Molecular Interactions in β-Lactoglobulin. III. Light
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Scattering Investigation of the Stoichiometry of the Association between pH 3.7 and 5.22.
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Journal of the American Chemical Society 82(12), 3168-3174.
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Zhou, P., Liu, X., Labuza, T.P., (2008). Effects of moisture-induced whey protein
465
aggregation on protein conformation, the state of water molecules, and the microstructure and
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texture of high-protein-containing matrix. Journal of Agricultural and Food Chemistry
467
56(12), 4534-4540.
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Zhu, H., Damodaran, S., (1994). Effects of calcium and magnesium ions on aggregation of
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whey protein isolate and its effect on foaming properties. Journal of Agricultural and Food
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Chemistry 42(4), 856-862.
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ACCEPTED MANUSCRIPT Table 1: Composition of four WPCs Sweet-WPC
Acid-WPC
Salty-WPC
Moisture (%)
5.5 ± 0.7ab
4.1 ± 0.2ac
5.7 ± 0.8b
3.9 ± 0.08c
Total protein (%)
84.1 ± 0.6a
61.8 ± 0.1b
67 ± 0.3c
10.9 ± 0.2d
Lactose (%)
0.1 ± 0.02a
2.9 ± 0.03b
0.001 ± 0.0001a
0.1 ± 0.03b
Ca (%)
0.04 ± 0.001a
0.02 ± 0.003b
Na (%)
0.01 ± 0.004a
0.4 ± 0.02a
0.03 ± 0.01b
1.1 ± 0.05b
6.5 ± 0.03a
6.4 ± 0.02b
4.2 ± 0.02c
5.5 ± 0.01d
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Lactic acid (%)
pH
0.7 ± 0.02c
2.4 ± 0.3c
0.2 ± 0.02c
0.07 ± 0.001b
0.14 ± 0.01c
0.08 ± 0.001c
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Values are means of at least 4 independent measurements (n ≥ 4); the results are presented as means ± standard deviation (SD). Different lowercase superscripts in the same row depict the significant difference between means for each spray dried whey powder.
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ACCEPTED MANUSCRIPT Table 2: Reaction rate, reaction order, rate constant (Kn) activation energy (Eo) and pre- and
476
pre-exponential factor (K0) of β-LG disappearance in different WPCs during storage
33
22 Sweet 33
22
Order of reaction
Kn
Eo
Ink0
((g Kg-1)(1-n) day-1)
(kJ mol-1)
((g Kg-1)(1-n) day-1)
4
0.09e
1.4a
7.0 x 10-5 a
25
0.18f
1.0b
1.2 x 10-3 b
45
0.29g
0.7c
1.5 x 10-2 c
4
0.11h
1.3d
1.4 x 10-4 d
25
0.15i
1.2e
4.7 x 10-4 e
45
0.39j
0.6f
3.9 x 10-2 f
4
0.13a
1.2g
2.8 x 10-4 g
25
0.22n
45
0.24o
4
0.16p
25
0.19l
45
0.24q
4 25 45
Salty
4 33
25
22 Acid 33
SEM a-x
0.9h
3.2 x 10-3 h
0.8i
6.9 x 10-3 i
1.1j
5.4 x 10-4 j
0.9k
2.6 x 10-3 k
0.8l
5.3 x 10-3 l
0.14b
1.2m
5.8 x 10-4 m
0.16k
1.1n
6.8 x 10-4 n
0.20l
1.0o
2.2 x 10-3 o
0.07d
1.7p
3.3 x 10-5 p
0.08m
1.6q
5.9 x 10-5 q
0.11h
1.4r
2.1 x 10-4 r
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Reaction rate for β-LG (µg / day)
96.3a
32.2a
97.6b
32.9b
58.3c
17.3c
41.2d
10.5d
23.1e
2.4e
31.9f
3.4f
13.2g
3.2g
54.5h
10.2h
0.1
0.04
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Temperature (°C)
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RH (%)
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4
0.12a
1.4s
1.3 x 10-4 s
25
0.13a
1.4t
1.9 x 10-4 p
45
0.14b
1.3u
2.8 x 10-4 t
4
0.06c
1.9v
1.7 x 10-6 u
25
0.06d
1.9w
4.5 x 10-6 v
45
0.10e
1.6x
3.8 x 10-5 w
0.001
0.0
1.4 x 10-3
Different lowercase superscripts in the same column depicts the significant differences between means of kinetic parameters. Results are expressed as means of two independent replications. SEM = Standard error of the mean.
477
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Table 3: Reaction rate, reaction order, rate constant (Kn) activation energy (E0) and pre-
479
exponential factor (K0) of α-LA disappearance in acid-WPCs during storage Reaction rate for α-LA (µg / day)
Order of reaction
Kn
E0
Ink0
((g Kg-1)(1-n) day-1)
(kJ mol-1)
((g Kg-1)(1-n) day-1)
4
0.15a
1.6a
9.4 x 10-5 a
25
0.19b
1.5b
1.9 x 10-4 b
45
0.20c
1.5c
3.9 x 10-4 c
4
0.13d
1.7d
6.4 x 10-5 d
25
0.17e
1.7e
8.7 x 10-5 e
45
0.18f
1.6f
1.7 x 10-4 f
0.001
0.0
7.9 x 10-4
Acid 33
SEM a-f
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Temperature (0C)
25.3 ± 0
1.7 ± 0
17.4 ± 0.1
2.2 ± 0.04
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ACCEPTED MANUSCRIPT List of Figures
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Figure 1: Vector normalized, smoothened and deconvoluted FTIR spectra of the Amide I
485
region (1600 – 1700 cm-1) for NWP over storage period of 0 (solid- black), 14 (long dashed-
486
black), 30 (short dashed- black), 60 (solid- grey) and 90 day (dashed- grey) under different
487
storage temperatures (4, 25 and 45 0C) and RH (22 and 33 %)
488
Figure 2: Vector normalized, smoothened and deconvoluted FTIR spectra of the Amide I
489
region (1600 – 1700 cm-1) for SWP over storage period of 0 (solid- black), 14 (long dashed-
490
black), 30 (short dashed- black), 60 (solid- grey) and 90 day (dashed- grey) under different
491
storage temperatures (4, 25 and 45 0C) and RH (22 and 33 %)
492
Figure 3: Vector normalized, smoothened and deconvoluted FTIR spectra of the Amide I
493
region (1600 – 1700 cm-1) for AWP over storage period of 0 (solid- black), 14 (long dashed-
494
black), 30 (short dashed- black), 60 (solid- grey) and 90 day (dashed- grey) under different
495
storage temperatures (4, 25 and 45 0C) and RH (22 and 33 %)
496
Figure 4: Vector normalized, smoothened and deconvoluted FTIR spectra of the Amide I
497
region (1600 – 1700 cm-1) for StWP over storage period of 0 (solid- black), 14 (long dashed-
498
black), 30 (short dashed- black), 60 (solid- grey) and 90 day (dashed- grey) under different
499
storage temperatures (4, 25 and 45 0C) and RH (22 and 33 %)
500
Figure 5: SDS Non-reduced PAGE patterns of whey protein powders at 0, 30 and 90 days of
501
storage. L1, L2, L3, L4 represent NWP, SWP, AWP and StWP under 22% RH, while L5, L6,
502
L7, L8 represent NWP, SWP, AWP and StWP under 33% RH, respectively. X1 represent
503
covalently linked aggregates with Mw ≥250 kDa. X2 represents medium size protein
504
aggregates with Mw~35 – 67 kDa. X3 represent β-LG A.
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Figure 6: SDS Reduced PAGE patterns of whey protein powders at 0, 30 and 90 days of
506
storage. L1, L2, L3, L4 represent NWP, SWP, AWP and StWP under 22% RH, while L5, L6,
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ACCEPTED MANUSCRIPT L7, L8 represent NWP, SWP, AWP and StWP under 33% RH, respectively. NDCA represent
508
non-disulphide covalently linked aggregates.
509
Figure 7: Changes in absolute quantity (µg) of non-reducible aggregates during storage of
510
NWP (solid line), SWP (dotted line), AWP (dashed line) and StWP (dashed-dotted line) at
511
storage temperatures of 4, 25 and 450C and relative humidity of 22 and 33%, as obtained
512
from image analysis of reducing SDS-PAGE.
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Figure 7:
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ACCEPTED MANUSCRIPT Highlights Properties of various WPCs were examined under different storage conditions.
•
β-LG was primarily involved in covalent cross linking in all WPCs.
•
Participation of α-LA in covalent crosslinking was evident primarily in acid-WPC
•
High storage temperature accelerated covalent linking between whey proteins
•
Reaction order of β-LG denaturation in acid and salty-WPCs was approximately 2
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