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Original Research

Frontiers in Soft Matter ·

Volume 6 - 2026 | https://doi.org/10.3389/frsfm.2026.1781355

Destabilizing foams of reconstituted acid whey powder at an elevated temperature using calcium-chelating salts

  • Nanik Purwanti1,2*corresponding author
  • Shane Mulcahy3
  • Eoin G. Murphy4*corresponding author
  1. 1Division of Biosystem Engineering, Faculty of Engineering and Technology, IPB University, Bogor, Indonesia
  2. 2Division of Chemical Engineering, Design and Smart Manufacture, Faculty of Engineering and Technology, IPB University, Bogor, Indonesia
  3. 3ArraTipp, Nenagh, Co. Tipperary, Ireland
  4. 4Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland
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Abstract

This study aimed to investigate the effects of calcium-chelating salts (CCS) namely trisodium citrate (TSC) and sodium hexametaphosphate (SHMP) on foamability and foam stability of acid whey powder (AWP) suspensions at 60 °C, which simulated the processing temperature of AWP in industry. Effects of salt concentrations, pH and suspended materials were observed. Foam capacity did not significantly change when pH was shifted from 6.8 to 4.6, when TSC or SHMP was added, or when centrifugation was applied to remove the suspended materials. In contrast, foam stability was substantially affected by these factors. Lowering pH and addition of CCS reduced foam stability, despite increasing protein solubility, in the case of CCS addition. This indicated protein aggregates or particulate structures in AWP suspensions play a dominant role in interfacial stabilization of the foams, which may be analogous to Pickering-like mechanisms. This was consistent with foam destabilization after complete removal of suspended materials. SHMP showed greater effectiveness in disrupting calcium-mediated aggregates than TSC, resulting in increased protein solubility. However, as illustrated by the Pickering-like behaviour mentioned above, increased protein solubility is not the only determining factor for improving foam stability in complex dairy systems. Nevertheless, SHMP offers a promising strategy to control excessive foaming during acid whey processing.

Keywords

1 Introduction

Whey proteins are well-known for their surface-active properties and are often applied as emulsifiers or foaming agents (Bals and Kulozik, 2003; Marinova et al., 2009; Alfaifi and Stathopoulos, 2010; Lazidis et al., 2016; Lajnaf et al., 2017; Du et al., 2021). In contrast to the many desirable foaming properties of whey proteins in food applications, foaming can also be a problematic occurrence during manufacturing of mineral acid whey (AW). Mineral AW is by-product of casein production, wherein milk is acidified by hydrochloric acid (HCl) to obtain a casein curd. AW is a complex colloidal system containing soluble proteins and a significant amount of minerals, particularly calcium and phosphorus which were released during the aforementioned acidification (Law and Leaver, 2000). The acidic conditions promote disruption of colloidal calcium phosphate and enhance protein-mineral interaction that led to formation of aggregated or particulate structures which may not fully dissolve (Law and Leaver, 2000; Joyce et al., 2018). Therefore, AW is a multiphase system containing both soluble and insoluble components. This intrinsic heterogeneity differentiates AW from purified whey systems, such as whey protein isolate (WPI) that are predominantly soluble. The physicochemical complexity of AW consequently can be expected to affect interfacial adsorption, foam formation and stability. In addition, the presence of non-dissolved components, including protein aggregates and insoluble calcium phosphate, may introduce particle-mediated stabilization or destabilization and altered drainage behaviour, which are not observed in WPI systems. Our previous research on AW foaming demonstrated significant occurrence of foaming during evaporation in an industrial production line, which further caused product overflow from tanks and associated process challenges. That study involved AW samples (∼20% total solids, pH 6.8) that were collected from AW processing plant. The experiments showed that AW at 61 °C had higher foam capacity and stability compared to AW at 21 °C. This observation highlighted the importance of temperature control during AW processing to counteract problematic foaming (Purwanti et al., 2022).

Foaming is commonly controlled using chemical antifoaming agents, such as silicone-based compounds, polyoxymethylene sorbitan esters, or fatty acid-based defoamers (European Union, 2008). These antifoaming agents act as surface-active additives under existing food additive regulation, i.e., Regulation (EC) No 1333/2008. However, increasing demand for clean-label dairy ingredients over artificial additives (Meijer, 2020) has driven the development of alternative physicochemical approaches. Among these approaches, calcium-chelating salts (CCS) have been proposed to modify protein interactions and reduce foam stability. Addition of calcium-chelating salts (CCS) was reported to increase heat stability of whey systems (Keowmaneechai and McClements, 2006; Hebishy et al., 2019; Scudeller et al., 2021). Zhu and Damodaran (1994a) demonstrated that WPI foams were the most stable with addition of 0.02–0.04 M Ca2+. Ca2+ at any concentration imparted higher foam stability than Mg2+. The role of Ca2+ in stabilizing foams was also observed in reconstituted low-heat skim milk powder (LHSMP) when foaming was performed at 50 °C (Kamath et al., 2011). Ibanoglu and Ibanoglu (1999) reported the highest foam stability in α-lactalbumin (α-la) was observed up to 0.2 wt% concentration in the presence of ethylenediaminetetraacetic acid (EDTA). This study suggested that adding EDTA to α-la solution may remove Ca2+ from the protein structure and lead to conformational changes and eventually, reduce surface tension of the α-la solution. EDTA dissociates casein micelles and increases casein in the milk serum, thus increases protein availability to stabilize air-water interface (Ward et al., 1997). However, the effects of EDTA on improved foam properties of milk was only observed until 10 mM. Further addition of EDTA up to 40 mM did not give further enhancement on foam properties. In contrast, addition of trisodium citrate (TSC) to the milk reduced its foam stability when foaming was performed at 45 °C and 65 °C (Kamath et al., 2011). Nevertheless, milk mostly contains casein which has completely different properties from whey. Therefore, casein’s behaviour towards EDTA or TSC cannot be directly related to the whey responses towards EDTA or TSC.

Other studies emphasized that pH plays a crucial role in foaming or the effectiveness of CCS (Phillips et al., 1990; Zhu and Damodaran, 1994b; Bals and Kulozik, 2003; Davis and Foegeding, 2004; Tosi et al., 2007; Lajnaf et al., 2018; Hu et al., 2019). Silva et al. (2013) described that demineralization of casein micelles by pH shifting from 6.8 to 5.5 did not change foam capacity but significantly reduced the foam stability. Shifting the pH from 6.8 to 5.5 reduced colloidal calcium phosphate, particle size, and concentration of sediments. Augustin and Clarke (2008) reported slightly different observation on LHSMP and high-heat skim milk powder (HHSMP). Shifting pH from 6.7 to 5.6 increased both foam capacity and stability, which was also explained by demineralisation of casein micelles. Addition of citric acid/TSC mixture or phosphate (N2HPO4) at 0.21 mol/kg LHSMP/HHMP increased foam capacity and stability of the milk at pH 5.6–7.9, but CaCl2 at the same concentration depressed foam capacity with foam stability only observed at pH 5.2 (Augustin and Clarke, 2008).

The above studies propose a somewhat contradictory and therefore, inconclusive mechanisms for the effect of chelating salts on modifying foaming properties of dairy systems, although few studies indicated the importance of Ca2+ removal to reduce foam stability of milk, especially at elevated temperatures (Kamath et al., 2011; Zhu and Damodaran, 1994b). The contrasting effects of CCS on foaming properties in milk systems might be attributed by the types of CCS used to modify salt equilibria and their concentrations (Silva et al., 2013). However, casein as the major component in milk might behave differently from whey. Furthermore, to the best knowledge of the authors, the effect of CCS on AW systems, which contain relatively large quantities of insoluble components, remains unexplored in the literature. Therefore, this work focused on investigating the effects of CCS at different concentrations, applied at neutral and acidic pH, at elevated temperature on foam stability of AW. Trisodium citrate (TSC) and sodium hexametaphosphate (SHMP) were applied to reconstituted AW powder because both salts are strong, food-grade Ca2+ sequestrants that are often applied in dairy system, especially caseins (Deshwal et al., 2023). This study sought to elucidate the mechanism through which TSC and SHMP modulate foaming properties of AW. This insight will facilitate AW processors to counteract foaming in their processing units. As Ca2+ has been known to promote protein-protein interaction, we hypothesize that CCS would disrupt calcium-mediated protein interactions in AW. This would be expected to reduce the presence of particulate structures and alter interfacial properties that govern foam stability.

2 Materials and methods

2.1 Materials

Acid whey powder (AWP) was provided by Arrabawn Co-Operative Society Ltd., Nenagh, Co. Tipperary, Ireland. Composition of the powder was 81% w/w lactose, 9.2% w/w protein, 8.4% ash, and 0.55% w/w fat (all in dry basis). Trisodium citrate (TSC) (Merck, Wicklow, Ireland) and sodium hexametaphosphate (SHMP) (Fischer Scientific, Dublin, Ireland) were used as calcium-chelating salts. Potassium hydroxide (KOH) and hydrochloric acid (HCl) (Merck, Wicklow, Ireland) were used for adjusting pH. Milli-Q water was used for dispersing the powder and diluting AWP suspension.

2.2 Methods

2.2.1 Preparation of acid whey powder suspensions and supernatants

Acid whey powder was slowly dispersed in pre-heated milli-Q water at 20% w/w total solids under continuous stirring at ∼500 rpm on a magnetic stirring plate to maximise dispersion during rehydration. The resultant suspension was allowed to hydrate under agitation for ∼16 h at room temperature (∼25 °C). TSC or SHMP was added to the suspension to achieve final salt concentrations of 10 or 20 mM; no salt was added to control suspensions. pH was adjusted to 6.8 using KOH or to 4.6 using HCl prior to foaming experiments. Additionally, centrifugation was applied to 20 mM TSC or SHMP AWP suspensions at pH 6.8 to study effects of insoluble materials on foaming, interfacial properties, and viscosity of the suspension. Approximately 30 mL of AWP suspension was poured in 50 mL centrifuge tubes and centrifuged at 16,000 × g for 10 min at 25 °C (Heraeus Multifuge X1R, F15-8x50cy rotor, Thermo Scientific, Osterode am Harz, Germany). The supernatant was carefully decanted and used for further experiments. The sediment was weighed and expressed as the weight percentage of sediment with respect to the initial weight of suspension. The protein content of the supernatant and sediment was analysed.

2.2.2 Calcium ion activity

Calcium ion activity was measured in freshly prepared AWP suspensions and supernatants of centrifuged AWP suspension using a combined ion-selective electrode (perfectION™ Ca2+ combination, Mettler Toledo, Dublin, Ireland). A standard calibration curve was set by diluting 1,000 mg/L calcium standard (Mettler Toledo, Dublin, Ireland) to 0.1, 1, and 10 mM. Calcium Ion Strength Adjuster (ISA) (perfectION™, Mettler Toledo, Dublin, Ireland), which mainly contains potassium chloride (KCl), was added into the standard solutions with a ratio of 1:50 and stirred for 1 min prior to immersing the electrode probe into the solution. The AWP suspension was prepared using Ca–ISA with the same procedures as previously described. Calcium ion activity was inferred from the electrode potential (mV) but the result was displayed as mM of Ca2+ concentration by automatic calibration against the standard calibration curve (Purwanti et al., 2024). Each determination was made in triplicate, and the results were presented as the average of Ca2+ concentration in mM.

2.2.3 Protein content

Crude protein content in AWP suspensions and supernatants was measured using a nitrogen analyser (Leco FP-628, Leco Corporation, Missouri, USA). This measurement was applied to samples with pH 6.8 containing 20 mM CCS and the reference samples. A nitrogen-to-protein conversion factor of 6.38 was applied and each sample was measured in triplicate.

2.2.4 Foam studies

Foam studies were performed on AWP suspensions and supernatants using Dynamic Foam Analyzer DFA100 (Krüss, Hamburg, Germany). Samples were pre-heated to 60 °C in a water bath set at 65 °C for 10 min which brought the sample temperature prior to foaming to 60 °C. This temperature was selected to mimic the conditions of an industrial AW evaporator, which differentiates this study from others carried out at room temperature. Samples (50 mL) were transferred immediately into a double-walled glass column (internal diameter of 40 mm) that had been set at 60 °C using a thermostatic water bath. This volume was defined through preliminary experiments to ensure that the liquid sample was completely foamed under experimental conditions. Temperature was equilibrated in the column for 5 min to compensate a temperature drop during sample transfer.

Foams were formed by compressed air at a flow rate of 0.8 L/min for 10 s through double filtered papers with diameter of 32 mm and pore size of 12–25 µm (FL4551, Krüss, Hamburg, Germany) mounted at the bottom of the column. The total height of foam and liquid was monitored continuously for 100 min by measuring transmission of blue light (λ = 469 nm) through the glass column. Optical contrast differences between the foam, liquid, and gas phases arising from blue light scattering at gas-liquid interfaces were used by Foam Analysis Software (ADVANCE, Krüss, Hamburg, Germany) to calculate the foam height and the height of liquid drained from the foams. Foam properties are distinguished as foamability and foam stability as described by Marinova et al. (2009) with the parameters tabulated in Table 1.

TABLE 1. Parameters for describing foamability and foam stability of AWP suspensions and supernatants.

Foam propertiesParametersEquation
FoamabilityFoam capacity (FC)(1)F C = V foam , 0 / V gas
Maximum foam density initially (MD 0)(2)M D 0 = V liq , 0 / V foam , 0
Foam stabilityFoam volume stability (FVS)(3)F V S = V foam / V foam , 0 × 100
Foam liquid stability (FLS)(4)F L S = V liq / V liq , 0 × 100
​Time at which the FLS has reduced to 25% of its initial value (t FLS25%)​​

V foam , 0 is the maximum foam volume initially, V foam and V foam , 100 is the foam volume remaining or 100 min after foam production, respectively, V gas is volume of the compressed air to produce foams, V liq , 0 is the initial liquid volume entrapped in foams, V liq and V liq , 100 is the remaining liquid volume entrapped in foams or 100 min after foam production, respectively.

DFA100 was equipped with foam structure module that allows 2-dimensional analysis of foam structures. The principal analysis of foam structures using the module was described by Oetjen et al. (2014). The results were presented as an average of initial radius of air bubbles and an average radius of air bubbles at t FLS25%.

2.2.5 Surface tension measurements

Dynamic surface tension, γ(t), was measured at 60 °C using an optical tensiometer (Attention Theta Flex, Biolin Scientific, Sweden) connected to OneAttention software v3.1. Pre-heated AWP suspensions or supernatants was prepared as described in Section 2.2.4. These samples were diluted 10x with milli-Q water, which gave a protein content of about 0.16 (±0.01) % w/w. pH of the diluted sample was adjusted to 6.8 or 4.6 and it was placed in a 1 mL syringe connected to a straight needle. A humidity-controlled chamber was mounted on the tensiometer’s sample stage and connected to a thermostatic water bath that maintained the internal chamber temperature at 60 °C. A syringe, filled with sample, was positioned inside the chamber and equilibrated for 15 min to stabilize the temperature. A drop of sample (∼8.5 µL) was produced, and the shape of the sample droplet was recorded continuously by a high-speed camera for 900 s. A measurement longer than this time was not possible because droplets typically detached from the needle.

The data of γ(t) as function of time was further analysed to obtain the rate of initial diffusion-controlled migration (k mig) which represents migration of the proteins from the bulk to the interface (Baeza et al., 2005; Niño et al., 2005), the rate of adsorption and unfolding (k a/u), and the rate of configurational re-arrangement of the proteins at the adsorbed layer (k arr) (Patino et al., 1999, 2007). Parameter k mig was obtained from the liner fit of π = γ 0 − γ t against t 1 / 2, where π is surface pressure (mNm-1) obtained from the difference between the surface tension in the absence of proteins (γ 0) and surface tension in the presence of proteins at time t (γ t). Parameters k a/u and k arr are first-order rate constant that are obtained from two linear regions of ln π s s − π t / π s s − π 0 against time (t), where π s s is the steady state surface pressure, π t is the surface pressure at time t, and π 0 is the initial surface pressure. The slope of the first linear region represents k a/u and the second slope represents k arr. More details of these steps are described elsewhere (Zhou et al., 2021; Purwanti et al., 2022).

2.2.6 Statistical analysis

Statistical differences among treatments were performed using SPSS. One-way analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) was performed at significant level of p < 0.05.

3 Results and discussions

3.1 Calcium ion activity

AWP suspensions at pH 6.8 originally contained 1.37 mM Ca2+, which was reduced by adding TSC or SHMP (Table 2) with SHMP having the stronger Ca2+ chelation effect of the two salts; [Ca2+] reduced by 66% and 77% with 10 mM and 20 mM SHMP, respectively, whereas the [Ca2+] reduction was 30% and 39% at corresponding TSC concentrations. This trend was consistent with other studies, which showed addition of SHMP to be a more effective Ca2+ chelator than (Prakash et al., 2007; Boumpa et al., 2008; Tsioulpas et al., 2010). [Ca2+] in AWP suspensions sharply increased by shifting the pH to 4.6; [Ca2+] is well documented to have an inverse relationship with pH (Tsioulpas et al., 2007; Chandrapala et al., 2015; Gaur et al., 2018). Addition of H+ by any means into milk or whey systems will increase [Ca2+] according to reaction (1) (Lewis, 2010). This equilibrium is very relevant for AWP suspensions because it contains considerable amounts of calcium and phosphate (Purwanti et al., 2023). About 50% of the Ca2+ in the AWP suspension with 20 mM TSC at pH 4.6 was chelated, whereas SHMP chelated up to 89% of the Ca2+ at the same concentration and pH, which were in line with other studies (De Kort et al., 2009, 2011; McCarthy et al., 2017).

3 Ca 2 + + 2 HP O 4 2 − ⇌ Ca 3 P O 4 2 + 2 H + (1)

TABLE 2. Effect of pH and calcium-chelating salts (CCS) on calcium ion concentration [Ca2+] of AWP suspensions and supernatants.

SamplepHCCS concentration (mM)[Ca2+] (mM)
TSCSHMP
​6.801.37 (±0.28)C1.37 (±0.28)C
Suspension100.96 (±0.06)C0.47 (±0.01)D
200.84 (±0.06)C0.31 (±0.02)D
4.6022.32 (±1.35)A22.32 (±1.35)A
2011.52 (±0.42)B2.52 (±0.13)B
Supernatant6.801.27 (±0.07)C1.27 (±0.07)C
200.90 (±0.02)C0.37 (±0.02)D

TSC, trisodium citrate, SHMP, sodium hexametaphosphate. Unequal capital letters within a column indicate the statistical difference (p < 0.05).

AWP suspensions at pH 6.8 contained considerable amount of sediment. Removing the sediment by centrifugation did not significantly reduce the [Ca2+], as shown in Table 2; [Ca2+] of the supernatant without added salts and with CCS added at 20 mM did not statistically differ from that corresponding AWP suspensions. The stability of [Ca2+] following centrifugation indicated that the measurement selectively targeted soluble Ca2+ fractions, as the removal of insoluble particles did not alter the analytical results.

3.2 Effects of CCS on AWP suspension

The presence of CCS affected the amount of sediment in pH 6.8 AWP suspensions. Significant reduction of sediment in the AWP suspension was observed with 20 mM TSC and SHMP (Figure 1). This observation can be attributed to calcium sequestration. TSC or SHMP binds Ca2+, thereby, disrupting calcium-phosphate bridges that stabilize protein aggregates in AW. Removal of calcium from protein complexes promotes dissociation of the aggregated protein, resulting in higher protein solubility and less sediment. This is in line with other studies which showed that addition of CCS had a correlation with reduction of sediment in dairy systems (Prakash et al., 2007; Sikand et al., 2016; McCarthy et al., 2017; Gaur et al., 2018; Hebishy et al., 2019; Purwanti et al., 2024). Addition of CCS dissociates Ca-containing sediment (Silva et al., 2013), from which the Ca2+ is subsequently chelated by the CCS. A previous study demonstrated that CCS induced dissociation of Ca-containing sediment was indicated by decreasing ash content of the sediment, along with increasing ash content in the supernatant (Purwanti et al., 2024).

Effects of CCS addition on the amount of sediment, expressed as the weight percentage of sediment with respect to the initial weight of the suspension, in AWP suspension pH 6.8. TSC – trisodium citrate, SHMP – sodium hexametaphosphate.
Figure 1. Effects of CCS addition on the amount of sediment, expressed as the weight percentage of sediment with respect to the initial weight of the suspension, in AWP suspension pH 6.8. TSC – trisodium citrate, SHMP – sodium hexametaphosphate.

This trend is in line with the observation on protein content in the sediment vs. supernatant as shown in Table 3. The protein content in the sediment reduced with addition of CCS. Conversely, the protein concentration in the supernatant increased. Table 3 implies that addition of CCS improves overall protein solubilization in AWP suspension, with 20 mM SHMP provided a significant effect, whereas TSC had no effect on protein content. Improvement of protein solubilization induced by SHMP might be attributed by its strong calcium chelating capacity. Phosphate group of SHMP binds calcium ions in 1:3 ratio (De Kort et al., 2009), whereas citrate binds calcium ions in 1:1 ratio (Liu et al., 2024).

TABLE 3. Effect of CCS addition on protein content (%w/w) of AWP suspension, supernatant, and sediment. Percentage of sedimented protein was calculated by taking the difference between the protein content in the suspension and supernatant.

CCSSuspension [1]Supernatant [2]Sediment [3]% Sedimented protein 1 − 2 / 1 × 100
0 mM1.76 (±0.04)B1.58 (±0.05)B3.44 (±0.15)A10.5
20 mM TSC1.73 (±0.02)B1.61 (±0.02)B3.26 (±0.27)A7.0
20 mM SHMP1.81 (±0.02)A1.71 (±0.04)A2.61 (±0.12)B5.6

Unequal capital letters indicate the statistical difference (p < 0.05) between applied CCS. TSC (trisodium citrate), SHMP (sodium hexametaphosphate).

3.3 Foamability parameters

Addition TSC or SHMP at the same concentration (20 mM) to WPI suspensions did not significantly change the maximum foam density (MD 0) and initial radius of air bubbles (r 0) (Table 4). Significant effect of pH on those parameters was also not observed. The only significant (p < 0.05) effect of CCS on foamability was observed in foam capacity (FC) when the pH was shifted from 6.8 to 4.6 with addition of 20 mM SHMP. This result was somewhat contradictory with other findings. Lajnaf et al. (2018) showed that raw acid bovine whey had a higher FC than raw sweet bovine whey because of the reduced electrostatic repulsion of proteins at acidic pH. Phillips et al. (1990) also showed that FC of 5% WPI was the highest at pH 5.0, followed by pH 7.0 and pH 4.0. Likewise, 0.1% solution of WPI or WPC exhibited the highest FC at pH ≈ 4.0 (Marinova et al., 2009). Marinova et al. (2009) suggested that the presence of precipitates in WPC solution reduce foamability of the solution, which might partially explain the results of this study. The AWP solutions either at pH 6.8 or 4.6, without or with addition of TSC/SHMP contained significant amount of suspended materials. Our previous experiment (using the same AWP) demonstrated that these materials were aggregates with average size (D4,3) of 41 μm (Purwanti et al., 2024). Addition of CCS decreased the size of these aggregates, the extent of which depended on CCS concentration. These suspended materials might be responsible in overruling the effects of pH and salt in foamability of AWP suspensions. The same reason may also be applicable for the maximum density of foams and initial radius of foams, which were similar between samples. Foegeding et al. (2006) emphasized that spontaneous adsorption of proteins from solution to air/water interface is the main importance for FC. This can be observed by reduction of interfacial/surface tension, which will be discussed later (see Section 3.5).

TABLE 4. Foam capacity (FC) and the maximum foam density (MD 0) of AWP suspensions, as well as the initial radius (r 0) of the foam bubbles. The parameters are listed as the functions of pH and salt types.

pHCCS (mM)FC (−)MD 0 (−)r 0 (μm)
6.8-01.85 (±0.05)A0.23 (±0.00)A126 (±1.41)AB
TSC201.78 (±0.02)BC0.23 (±0.00)A119.5 (±3.54)B
SHMP201.80 (±0.02)ABC0.23 (±0.00)A122 (±0.00)B
4.6-01.78 (±0.01)ABC0.23 (±0.02)A138 (±8.49)A
TSC201.84 (±0.02)AB0.22 (±0.00)A127.5 (±4.95)AB
SHMP201.78 (±0.01)C0.23 (±0.00)A126 (±11.31)AB

Unequal capital letters within a column indicate the statistical difference (p < 0.05) between different pH and CCS.

Similar trends of FC, MD 0, and r 0 as those previously described in Table 4 are also shown in Table 5, which highlights the effects of salt concentrations. There are no substantial differences observed on foamability parameters when TSC or SHMP was added to the AWP suspensions at different concentrations.

TABLE 5. Foam capacity (FC) and the maximum foam density (MD 0) of AWP suspensions at pH 6.8, as well as the initial radius (r 0) of the foam bubbles. The parameters are listed as the function of salt types and concentrations.

CCS (mM)FC (−)MD 0 (−)r *0* (μm)
-01.85 (±0.05)A0.23 (±0.00)A126 (±1.41)AB
TSC101.82 (±0.08)A0.24 (±0.01)A128 (±5.66)A
​201.78 (±0.02)A0.23 (±0.00)A119.5 (±3.54)B
SHMP101.85 (±0.03)A0.23 (±0.02)A119 (±2.83)B
​201.80 (±0.02)A0.23 (±0.00)A122 (±0.00)AB

Unequal capital letters within a column indicate the statistical difference (p < 0.05) between different salts and concentrations.

Removing the suspended materials by centrifugation prior to foaming experiments led to more varied foamability parameters (Table 6). Without salt, FC of the supernatant was significantly lower than the suspension. However, when 20 mM of either CCS was added, there was no significant difference between suspensions and supernatants. Foams were significantly denser when the suspended materials were removed, as shown by MD 0 values. This indicates more continuous phase entrapped within the lamellae and Plateau borders, resulting in an increased liquid hold-up (Stone et al., 2003).

TABLE 6. Foam capacity (FC) and the maximum foam density (MD 0) of AWP suspensions and supernatants at pH 6.8, as well as the initial radius (r 0) of the foam bubbles. The parameters are listed as the function of salt types at the same concentration.

CCS (mM)FC (−)MD 0 (−)r *0* (μm)
-0, susp*.1.85 (±0.05)A0.23 (±0.00)A126 (±1.41)A
0, sp.1.76 (±0.05)B0.21 (±0.02)AB113 (±11.31)A
TSC20, susp*.1.78 (±0.02)AB0.23 (±0.00)A119.5 (±3.54)A
20, sp1.80 (±0.00)AB0.20 (±0.00)B114 (±5.66)A
SHMP20, susp*.1.80 (±0.02)AB0.23 (±0.00)A122 (±0.00)A
20, sp.1.79 (±0.01)AB0.20 (±0.00)B116 (±5.66)A

susp* represents AWP, suspension, the data are the same as those in Table 5. They are placed again here for the reader’s convenience in making direct comparison with the supernatant. Unequal letters within a column indicate the statistical difference (p < 0.05).

3.4 Foamability stability and structure

3.4.1 Effects of pH and salt types

Despite having no effect on initial foam properties (FC, MD 0, and r0), pH and CCS type did affect foam destabilization. Foam stability is indicated by foam volume stability (FVS) and foam liquid stability (FLS). FVS of the AWP suspension without salt at pH 6.8 after 100 min was significantly higher than that of the suspension at pH 4.6, indicated more stable foams at pH 6.8 (Table 6). Whey protein typically exhibits an isoelectric point at around pH 5.0–5.3; therefore, AWP suspensions at pH 6.8 are predominantly negatively charged, whereas they are expected to carry a net positive charge at pH 4.6. Our previous study demonstrated that AWP suspension at pH around 6.0 without CCS had zeta potential of −12.35 mV at room temperature (Purwanti et al., 2024). In that study, zeta potential was affected by CCS addition, and varied with the type of CCS used, concentrations, and temperature. At acidic pH, close to the iso-electric point of whey protein, proteins carry almost no net charges and might cause less electrostatic stabilization of bubbles, leading coalescence and unstable foams (Dickinson, 2017). However, the zeta-potential of at pH 4.6 was not measured in this study and as mentioned above is likely to be highly dependent on CCS type and concentration. Therefore, it is likely that the foam destabilization in a complex systems like AWP suspensions cannot be attributed solely to electrostatic interactions. Destabilization likely reflects concurrent changes in aggregate structure and interfacial behaviour with the change of pH.

FVS of AWP suspensions decreased significantly when TSC or SHMP was added. Adding CCS sequesters Ca2+ that may bridge negatively charged groups of adjacent protein molecules (Havea et al., 2002). The presence of these protein molecules at air-water interface with reduced Ca2+ might weaken the interfacial films, leading foams to coalesce faster. Previous research reported that α-lactalbumin, one of whey protein fractions, became more surface active when losing its bound Ca2+ (Zhang et al., 2004). Table 7 indicates that the role of pH was overruled when CCS was added. FVS values were not significantly different between pH 6.8 and 4.6 with TSC or SHMP added. Marinova et al. (2009) and Lajnaf et al. (2018) have shown pH-dependence of whey foam stability putatively caused by the effect of different surface charges on protein interactions. This can also be observed in this study through a significant change of FVS when pH of the AWP suspension without CCS shifted from 6.8 to 4.6. However, the pH shift from pH 6.8 to 4.6 with added CCS might not dramatically change conformation or interfacial properties of the mixed whey system. This observation suggests that disruption of calcium-mediated aggregation and the resulting particulate structures play a more dominant role than electrostatic interaction in determining foam stability.

TABLE 7. Foam stability parameters of AWP suspensions at pH 6.8 and 4.6 as functions of pH and salt types at the same concentration.

pHCCS (mM)FVS (%) at 100 minFLS (%) at 100 min
6.8-048.30 (±1.27)A16.30 (±0.71)AB
TSC2033.40 (±2.55)BC15.00 (±1.13)BC
SHMP2017.35 (±4.45)D13.05 (±1.48)C
4.6-037.25 (±1.06)B18.05 (±0.49)A
TSC2030.05 (±4.45)C16.40 (±1.27)AB
SHMP2019.10 (±1.27)D13.10 (±0.71)C

Unequal capital letters within a column indicate the statistical difference (p < 0.05).

The liquid volume that remained entrapped in the foams, indicated by FLS, had different trend from FVS. FLS was not affected by pH shift nor TSC addition, but it was significantly affected by 20 mM SHMP addition. At pH 4.6, solubility of proteins decreased and aggregation increased. Aggregates were expected to form thicker films (Krešic et al., 2006) which entrapped water in foam lamella, leading to longer time for liquid to drain. However, this was not the case. The liquid drainage in protein foams might be governed by a complex interaction between interfacial phenomena at the bubble scale and collective transport processes throughout foam networks. Consequently, changes in protein aggregation or interfacial film properties might be offset by foam geometry, bubble packing, and liquid flow through Plateau borders, leading to comparable FLS despite differences in molecular state, which is consistent with the study of Audebert et al. (2019). Braunschweig et al. (2016) demonstrated that protein aggregates tend to increase stability of foam films when [Ca2+] originated from CaCl2 of more than 30 mM was added. Nevertheless, the highest foam stability at high [Ca2+] was exhibited macroscopically by much smaller bubble sizes. In this research, an increase in foam stability was observed with 20 mM SHMP, but not with 20 mM TSC. This indicates that mild calcium sequestration was insufficient to alter foam drainage, whereas strong chelation combined with polyphosphate-protein interactions might modify interfacial and structural properties of the foam, leading to more stable foams.

Initial foam structures in CCS-free suspensions were significantly affected by pH. Larger bubbles were observed at pH 4.6. The structures progressively altered over time as shown in Figure 2. A significant difference was observed between the structures at t 0 and t FLS25% as the bubbles collapsed or coalesced forming larger bubbles over time. A difference was also observed between foam structures without and with CCS at the same pH; CCS addition resulted in smaller bubbles. However, no substantial difference was observed between TSC and SHMP at the same pH.

Foam structures made of AWP suspensions at the time the foaming was stopped or *t*0 (left panel: **(A, C, E, G, I, K)**), and at the time when 75% of the entrapped liquid drained or *t*FLS25% (right panel: **(B, D, F, H, J, K)**). The white scale represents 2 mm.
Figure 2. Foam structures made of AWP suspensions at the time the foaming was stopped or t0 (left panel: (A, C, E, G, I, K)), and at the time when 75% of the entrapped liquid drained or tFLS25% (right panel: (B, D, F, H, J, K)). The white scale represents 2 mm.

3.4.2 Effects of salt types and concentrations

Table 8 shows that adding 10 mM TSC to AWP suspension did not significantly reduce FVS and FLS at 100 min after foam production. However, 20 mM TSC significantly reduced FVS although FLS remained the same as that with 10 mM TSC. Adding 20 mM TSC might weaken gas-liquid interface, leading to bubble coalescence or shrinkage, without affecting the liquid hold in the foam’s lamellae. A different trend was observed with addition of SHMP to AWP suspension; 10 mM SHMP significantly reduced FVS at 100 min. This observation might be linked to the fact that SHMP is stronger Ca2+ chelator than TSC. Sequestered Ca2+ from AWP suspension would alter interfacial properties, leading to foam destabilization mainly due to droplet shrinkage because FLS value insignificantly affected.

TABLE 8. Concentration effects trisodium citrate (TSC) and sodium hexametaphosphate (SHMP) on foam volume stability (FVS) and foam liquid stability (FLS) at 100 min after foam production.

CCS (mM)FVS (%) at 100 minFLS (%) at 100 min
-048.30 (±1.27)A16.30 (±0.71)A
TSC1044.00 (±4.81)A14.55 (±0.49)AB
2033.40 (±2.55)B15.00 (±1.13)AB
SHMP1030.20 (±5.80)B12.80 (±0.14)B
2017.35 (±4.45)C13.05 (±1.48)B

Unequal capital letters within a column indicate the statistical difference (p < 0.05).

3.4.3 Effects of sediment

Overall, removal of suspended materials from AWP suspensions at pH 6.8 largely resulted in substantial differences on FVS, FLS, and foam structures. Visually, foam bubble populations were markedly larger in samples containing suspended materials (Figure 2) than the populations in which suspended materials were absent (Figure 3). Comparing Figures 2, 3 underscores the importance of suspended materials in controlling foam destabilization due to liquid drainage. FVS of AWP supernatant at 100 min without salt was 7.66%, dropped about 84% compared with AWP suspension. At the presence of particles, the liquid phase might create viscous lamellae and Plateau borders. In this case, Zou et al. (2024) demonstrated that the size of bubbles remained unchanged for 30 days after foaming. The role of particles in foam stability flips the known trend that drainage velocity of liquid through Plateau borders is proportional to the square of bubble radius (U ≈ r 2), therefore the drainage time is inversely proportional to the square of the radius (t ≈ 1/r 2) (Saint-Jalmes and Langevin, 2002). The observation on the effect of sediment confirms that high stability of foam produced by the AWP suspensions resulted from the adsorption of insoluble fractions at the air-water interface, creating a rigid physical barrier.

Foam structures made of AWP supernatants at the time the foaming was stopped or *t*0 (left panel: **(A,C,E)**), and at the time when 75% of the entrapped liquid drained or **t**FLS25% (right panel: **(B,D,F)**). The white scale represents 2 mm.
Figure 3. Foam structures made of AWP supernatants at the time the foaming was stopped or t0 (left panel: (A,C,E)), and at the time when 75% of the entrapped liquid drained or tFLS25% (right panel: (B,D,F)). The white scale represents 2 mm.

Once the suspended materials were removed, the effect of TSC or SHMP at the same concentration in liquid drainage was overruled. The significant difference of FLS values at 100 min after removal of the suspended materials were only observed in AWP without CCS added. This confirmed less pronounced effect of TSC or SHMP in liquid drainage of AWP foams, as observed in bubble sizes at t FLS25%. Several studies have shown that salts may slow down liquid drainage from foams, but this depended on salt types and concentrations as well as polymers or surfactants that stabilize the foams (Micheau et al., 2013; Obisesan et al., 2021; Xiao et al., 2022). In contrast to FLS, FVS at 100 min dropped about 72% with TSC and 28% with SHMP when the suspended materials were removed. This result indicates that the presence of CCS remained crucial in foam destabilization but through foam coalescence. However, TSC or SHMP did not show significant difference in this matter because the average bubble radius between the supernatant with added TSC or SHMP was similar, meaning that coalescence might occur at the same rate.

3.5 Surface properties

Figure 4 showed the interfacial tension dynamics of AWP suspensions at pH 6.8 and 4.6, with or without CCS, and the corresponding supernatant obtained after centrifugation at pH 6.8. Interfacial tension of the suspension at pH 6.8 (round black symbol) decreased over time. The reduction was observed when 20 mM TSC or SHMP was added. At the beginning, SHMP led to further reduction of the interfacial tension compared to TSC; However, the reduction slowed down towards the end of measurement, resulting in higher interfacial tension of AWP suspensions containing SHMP compared to TSC. Reducing the pH to 4.6 resulted in much lower interfacial tension of the suspension than those at pH 6.8 with or without TSC. The suspension with 20 mM TSC at pH 4.6 had similar trend of interfacial tension reduction as the suspension without salt until about 200 s, after which a significant reduction was observed. On the contrary, the interfacial tension of the suspension with 20 mM SHMP at pH 4.6 was not reduced. Instead, the interfacial tension of the suspension was higher than the suspension without salt. The observation that addition of 20 mM SHMP to AWP suspensions at pH 6.8 or 4.6 did not reduce the interfacial tension contradicts the conventional expectation that higher protein solubility would lower surface tension, thereby increase surface activity. The observation in this study is consistent with previous studies indicating that high concentration of soluble proteins does not necessarily increase the surface activity (Dickinson, 2010). The surface activity in complex protein systems is determined by the combined effects of protein adsorption, intermolecular interactions, and the presence of particulate structures. These structures may form rigid, jammed layers at the interface that resist deformation and drainage (Murray, 2007). Those previous studies also align with the behaviour of centrifuged AWP suspensions observed in the current study. The supernatants showed consistently higher dynamic interfacial tension, even in the presence of TSC and SHMP, compared to the corresponding suspensions (Figure 4). The observations shown in Figure 4 could indicate that calcium sequestration weakens the interfacial layers although protein solubility increases. To explain this unexpected behaviour, protein adsorption dynamics at the interface were further analysed.

Dynamic interfacial tension of AWP suspensions at pH 6.8 and 4.6, and the corresponding supernatant at pH 6.8, measured over 900 s. TSC – trisodium citrate, SHMP – sodium hexametaphosphate, Sp* – supernatant.
Figure 4. Dynamic interfacial tension of AWP suspensions at pH 6.8 and 4.6, and the corresponding supernatant at pH 6.8, measured over 900 s. TSC – trisodium citrate, SHMP – sodium hexametaphosphate, Sp* – supernatant.

Foamability and foam stability of proteins are inherently linked to the interfacial properties (Buchmann et al., 2019). Three parameters can be used to describe protein dynamics at the interfaces, i.e., the rate of initial diffusion-controlled migration (k mig), the rate of adsorption and unfolding (k a/u), and the rate of configurational re-arrangement of the proteins at the adsorbed layer (k arr), all of which can be derived from dynamic interfacial tension data. The value k mig is derived from the initial slope of π against t 1/2 and a good fit can be obtained if the π values are low (<10 mN/m) (Zhou et al., 2021). However in this study, the initial values of π were larger than 10 mN/m, indicating that migration of AWP from the bulk to the interface was very fast (Perez et al., 2012). This seems to be the case for particles or mixed whey systems. Peng et al. (2017) showed that gliadin particles with size of hundreds of micrometers had a faster mass transport rate than soy glycinin, β-lactoglobulin, and casein. Therefore, k mig in this study was still evaluated to get rough estimation of how migration of AWP differed between treatments. Table 9 shows that the rate of diffusion-controlled migration (k mig) of AWP from the bulk to the air-suspension interface at pH 6.8 significantly decreased when 10 mM TSC or SHMP was added. In contrast, a significant increase of k mig was observed at pH 4.6 when 20 mM SHMP was added. Without CCS presence, the pH shift from 6.8 to 4.6 also significantly increased the k mig. Peng et al. (2018) also observed similar trend in gliadin particle where migration rate depends on the balance between interparticle interactions and dispersion state. At pH 4.6, which is closer to the isoelectric region of whey systems, changes in intermolecular interactions in AWP suspensions may reduce barriers to transport and facilitate rapid migration of proteins. However, this effect is more likely associated with alterations in aggregate structures and interaction force rather than electrostatic effects alone. At 20 mM, SHMP appeared to facilitate protein migration, whereas TSC remained ineffective. At lower concentration, i.e., 10 mM, both salts may promote intermediate destabilization (Dickinson, 2010), resulting in reduced migration. The rate of migration of AWP without suspended materials (supernatant) at pH 6.8 did not differ significantly from AWP suspension at the same pH. Adding 20 mM TSC did not change the k mig but 20 mM SHMP significantly decreased the k mig. This is a contrasting observation from k mig with 20 mM SHMP at pH 4.6. The effect of SHMP on k mig seemed system dependent. SHMP likely promoted protein-polyphosphate association in the particle-free system at pH 6.8, resulting in reduced mobility. In contrast, at pH 4.6 and in the presence of suspended materials, SHMP acted as a strong chelating agent that may disrupt weakly stabilized proteins, thereby, accelerating migration.

TABLE 9. Characteristic parameters for the rate of migration (k mig), the rate of adsorption and unfolding (k a/u), and the rate of re-arrangement (k arr) of AWP suspension or its supernatant at the air/water interface, without or with the presence of trisodium citrate (TSC) or sodium hexametaphosphate (SHMP).

pHCCS (mM)TSCSHMPTSCSHMPTSCSHMP
k mig (mNm−1s−0.5)k a/u × 103 (s−1)k arr × 103 (s−1)
6.8037.78 (±0.66)A,a2.53 (±0.95)A,a13.5 (0.74)A,a
1036.87 (±0.87)B35.66 (±0.99)b2.38 (±0.97)A2.87 (±0.97)a13.1 (±0.64)A15.3 (±0.72)a
2037.59 (±0.41)AB37.44 (±1.64)a2.73 (±0.88)A3.23 (±0.97)a18.7 (±0.66)A16.2 (±0.68)a
4.6039.94 (±0.67)A,b5.30 (±0.92)A,a-
2040.50 (±1.35)A41.32 (±0.38)a1.93 (±0.83)B1.92 (±0.92)b5.47 (±0.79)17.3 (±0.60)
Sp*, 6.8036.85 (±1.03)A,a3.18 (±0.96)A,a12.9 (±0.52)A,a
2036.16 (±1.35)A35.44 (±0.55)b2.57 (0.97)B2.48 (0.95)b15.8 (0.81)A18.9 (0.76)a

Sp* is supernatant, unequal capital letters in a column indicate significant difference between TSC treatments, whereas unequal lowercase letter indicate significant difference between SHMP treatments.

After reaching the interface, the partially unfolded proteins adsorbed and strongly anchored to the interface at similar rates in AWP suspensions with or without salts at pH 6.8 (Table 9). The k a/u implies a characteristic time (τ = 1 / k) of ∼5–7 min for the proteins to penetrate the interface, anchoring, and undergo initial conformational changes. This characteristic time decreased to about 3 min when pH of the suspension without salt was shifted to 4.6. However, addition of TSC or SHMP at this pH slowed down the process significantly, indicated by τ of around 8 min. Removing suspended materials (supernatant) resulted in k a/u values similar to those of suspensions, which emphasized the domination of soluble proteins at the interfacial activities or the absence of particle interference during the measurement because of dilution effect or solid precipitation.

The k arr values (Table 9) indicated that the proteins re-arranged quickly once anchored at the interface. The characteristic time of this process in AWP suspensions at pH 6.8 with or without salts was not significantly different (53–76 s). This indicates that the rate-limiting factor of interfacial behaviour of the AWP was adsorption and unfolding. The k arr value of AWP suspension at pH 4.6 without added CCS cannot be identified because all data points fit into regression linear of k a/u. A longer measurement of dynamic interfacial tension should have been performed to capture k arr value, but this was not possible technically because of unstable droplet over 900 s at 60 °C. Addition of TSC at pH 4.6 significantly decreased k arr, that implies longer time was required for re-arrangement of the proteins at the interface. Citrate might increase the kinetic barrier for spreading and slowing relaxation of the adsorbed proteins because it promotes tighter interactions and earlier interfacial crowding through ionic-strength effect and moderate calcium sequestration. In contrast, stronger chelation by SHMP may limit interfacial jamming and maintain lateral mobility. Consequently, the characteristic re-arrangement time is not prolonged in the presence of SHMP.

The above suggests two roles of AWP in foamability and foam stability of AWP foams. First, soluble proteins were responsible for the interfacial behaviour (Table 9), governing foamability because a fast rate of initial adsorption is required (Fameau et al., 2023). Therefore, differences were not observed in foam capacity between AWP suspension and supernatant (Table 6). The presence of suspended materials also affects interfacial properties but in a different way to soluble proteins. The suspended materials may create a jamming network at the interface, leading to high viscoelastic interface that hinders liquid drainage and gas diffusion (Binks, 2002; Fameau et al., 2023). Therefore, foam stability in terms of FVS and FLS was higher in AWP suspensions compared to supernatant. In acid whey systems, calcium-mediated interactions promote formation of aggregates that can adsorb at the air-water interface and contribute to foam stabilization through a Pickering-like mechanism. While suspended materials, pH, and salt addition (types and concentrations) did not affect foamability of AWP suspensions, they significantly affected foam stability. Removing the suspended materials, reducing pH and adding TSC or SHMP decreased foam stability. Foam stability, in principle, depends on the ability of the adsorbed layer to develop a viscoelastic network at the interface that resists drainage and coalescence. Therefore, interfacial rheology analysis would be beneficial in the future to confirm the mechanical properties of the adsorbed layer and further substantiate the proposed mechanisms. This would complement dynamic interfacial tension analysis that primarily captures adsorption kinetics and interfacial coverage.

Destabilization mechanism induced by either TSC or SHMP can be attributed to calcium sequestration and the consequent disruption of protein aggregates. In this study, SHMP demonstrated as a stronger foam stability reductor than TSC. This is likely attributed to higher calcium-binding affinity, which was also confirmed by the data in Table 2, and its greater effectiveness in disrupting calcium-mediated aggregates, which was confirmed by the data in Table 3 and Figure 1. Although soluble protein fraction increases significantly with SHMP, the soluble proteins alone are insufficient to form a sufficiently viscoelastic film at 60 °C to compensate for the loss of the particulate barrier, leading to foam collapse.

4 Conclusion

This study demonstrated that foam properties of AWP suspensions, which is a complex colloidal system containing high mineral content, soluble and insoluble components, are strongly system-dependent and cannot be explained solely by calcium sequestration. The particulate structures in the form of protein aggregates, insoluble calcium phosphate, or other suspended materials, which are often considered undesirable in many processing contexts, showed a dominant role as particulate stabilizers at the air-water interface that contributes to foam stability. The addition of CCS disrupted protein aggregates, increased protein solubility, reduced the amount of sediment in AWP suspensions, but also removed the particle-mediated stabilization at the interface. This mechanism was supported by the observation that complete removal of the sediment by centrifugation resulted in increased interfacial tension and decreased foam stability. This demonstrates that enhanced protein solubility does not necessarily improve interfacial properties in complex systems. In this case, removing the structural sediments contributed to destabilize AWP foams.

Comparing TSC to SHMP, addition of SHMP had a more pronounced effect in destabilizing AWP foams. Therefore, SHMP could be an alternative to destabilize foams during AWP processing at 60 °C. Nevertheless, the specific effects of SHMP on AWP varied with pH and the presence of suspended materials. These varied effects of the same salt highlight the existence of distinct physicochemical regimes, which might worth further study. Overall, the findings underscore that foamability, foam stability, and interfacial re-arrangement in AWP suspensions are governed by a complex interplay between chelator strength, protein charge state, interfacial mobility, and bulk constraints, rather than from single factor only.

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Footnotes

Edited by: Thomas A. Vilgis, Max Planck Institute for Polymer Research, Germany

Reviewed by: Clémence Le Coeur, Université Paris-Est Créteil Val de Marne, France

Siti Ari Budhiyanti, Gadjah Mada University, Indonesia

Statements

Conflict of interest

Author SM was employed by ArraTipp.

Author EGM was employed by Teagasc.

The remaining author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

The author(s) declared that financial support was received for this work and/or its publication. The authors acknowledge the financial support of Enterprise Ireland under the grant number MF20180049 and the EU Framework Programme for Research and Innovation Marie Sklodowska-Curie Actions, grant number 713654.

Data availability

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Author contributions

NP: Writing – original draft, Formal Analysis, Writing – review and editing, Methodology, Investigation. SM: Conceptualization, Writing – review and editing, Resources. EGM: Resources, Funding acquisition, Project administration, Validation, Writing – review and editing, Supervision.

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Citation

Purwanti, N., et al. (2026). Destabilizing foams of reconstituted acid whey powder at an elevated temperature using calcium-chelating salts. Frontiers in Soft Matter, Volume 6. https://doi.org/
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6 - 2026

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Copyright © 2026 Purwanti, Mulcahy and Murphy.

License: https://creativecommons.org/licenses/by/4.0/

*Correspondence: Nanik Purwanti; Eoin G. Murphy

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