Rheological and Solubility Properties of Soy Protein Isolate

molecules

Article Rheological and Solubility Properties of Soy Protein Isolate

Timothy D. O’Flynn 1,2, Sean A. Hogan 1 , David F. M. Daly 3, James A. O’Mahony 2 and Noel A. McCarthy 1,*

1 Teagasc Food Research Centre, Food Chemistry & Technology Department, Moorepark, Fermoy, P61 C996 Cork, Ireland; timothy.oﬂ[email protected] (T.D.O.); [email protected] (S.A.H.) 2 School of Food and Nutritional Sciences, University College Cork, T12 YT20 Cork, Ireland; [email protected] 3 Abbott Nutrition, a Division of Abbott Laboratories, Cootehill, H16 RK50 Cavan, Ireland; [email protected] * Correspondence: [email protected]; Tel.: +353-(0)25-42202

Abstract: Soy protein isolate (SPI) powders often have poor water solubility, particularly at pH values close to neutral, which is an attribute that is an issue for its incorporation into complex nutritional systems. Therefore, the objective of this study was to improve SPI solubility while maintaining low viscosity. Thus, the intention was to examine the solubility and rheological properties of a commercial SPI powder at pH values of 2.0, 6.9, and 9.0, and determine if heat treatment at acidic or alkaline conditions might positively influence protein solubility, once re-adjusted back to pH 6.9. Adjusting the pH of SPI dispersions from pH 6.9 to 2.0 or 9.0 led to an increase in protein solubility with a concomitant increase in viscosity at 20 ◦C. Meanwhile, heat treatment at 90 ◦C significantly improved the solubility at all pH values and resulted in a decrease in viscosity in samples heated at pH 9.0. All SPI dispersions measured under low-amplitude rheological conditions showed elastic- 0 like behaviour (i.e., G > G”), indicating a weak “gel-like” structure at frequencies less than 10 Hz. In summary, the physical properties of SPI can be manipulated through heat treatment under acidic Citation: O’Flynn, T.D.; Hogan, S.A.; or alkaline conditions when the protein subunits are dissociated, before re-adjusting to pH 6.9. Daly, D.F.M.; O’Mahony, J.A.; McCarthy, N.A. Rheological and Keywords: soy protein; thermal treatment; pH; solubility; rheology Solubility Properties of Soy Protein Isolate. Molecules 2021, 26, 3015. https://doi.org/10.3390/ molecules26103015 1. Introduction According to the USDA, 360 million metric tons of soybean was produced globally Academic Editor: Eugenio Aprea in 2018, ranking it the highest volume oilseed crop in the world [1]. An excellent source of bioavailable protein, soybeans are composed of ≈40% protein, 18–22% oil, and 35% Received: 19 April 2021 carbohydrate [2–4]. Having good amino acid quality, with the exception of methionine, Accepted: 14 May 2021 Published: 19 May 2021 soy protein is often used as a major protein ingredient source in food product formu- lation [5]. There are four major soybean storage proteins; 2S, 7S, 11S, and 15S, two of β Publisher’s Note: MDPI stays neutral which, 7S ( -conglycinin) and 11S (glycinin), are responsible for up to 35 and 52% of the with regard to jurisdictional claims in total protein content, respectively [6,7]. These storage proteins are shown to be consumed published maps and institutional affil- rapidly during soybean germination for their nitrogen component [8]. Glycinin proteins iations. are hexamers, with molecular masses in the range 320 to 375 kDa, and they are composed of two trimers, containing a number of polypeptide chains linked via disulphide bonds [9]. The β-conglycinin proteins are composed of three homologous proteins with a molecular weight of 76, 72, and 53 kDa and are held together via non-covalent interactions [9]. Soy protein concentrates (SPC) and isolates (SPI) are typically produced through a Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. combination of dry (dehulling and milling) and wet (aqueous alcohol washing and acid This article is an open access article precipitation) processing techniques [10]. SPIs generally have a protein content greater distributed under the terms and than 90%, w/w, and are used extensively in nutritional formulations due to their high conditions of the Creative Commons protein content, neutral taste, and excellent emulsification properties [8,11–13]. However, Attribution (CC BY) license (https:// one of the main limiting factors with the utilisation of soy protein is its inherent low creativecommons.org/licenses/by/ solubility [14]. The solubility of SPI is influenced by multiple factors, such as dissolution 4.0/). temperature, pH, and ionic strength [15], with lowest solubility (≈10%) at pH 4.6 (isoelectric

Molecules 2021, 26, 3015. https://doi.org/10.3390/molecules26103015 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 3015 2 of 15

point) [16]. Lokuruka (2011) stated that thermal treatment and solvent-based approaches used for protein extraction may influence solubility by denaturing proteins and exposing hydrophobic amino acid residues [17]. Although the solubility of soy protein may not be as high as some animal proteins such as whey, the solubility profile of soy protein allows it to be a good protein ingredient source [18]. The influence of pH on solubility of soy protein has been shown to display a U-shaped profile in which the highest solubility of SPI is achieved on either side of the isoelectric point (pI ≈ 4.6), with complete protein solubility potentially achieved at pH 11 [19]. While Nakai et al. (1980) showed that through the addition of sodium dodecyl sulphate (SDS) and alterations of pH (10% SDS at pH 12, 22 ◦C for 5 min), the solubility of SPI can be increased by up to 24% [20]. The interaction between hydrophobic groups within the SDS molecule and the hydrophobic amino acid residues on the soy proteins result in an increase in the negative charge of the protein, preventing aggregation and subsequent precipitation. However, simple adjustment of pH to highly acidic or alkaline environments causes a dissociation of the complex protein structure of β-conglycinin and glycinin into their representative subunits, resulting in a significant increase in protein solubility [21,22]. It has also been shown, for a variety of different commercial SPI ingredients, that greater solubility is achieved in heated systems containing higher levels of low molecular weight subunits (10–400 KDa) compared to those containing higher molecular weight (>400 KDa) material [23]. Lee et al. (2003) showed that the solubility of SPI in 0.1 M NaCl at 25 ◦C did not vary significantly over a range of thermal heat treatment temperatures (i.e., ranging from 25 to 75 ◦C) [15]. A reason for this may be related to the fact that the temperature range used did not exceed the denaturation temperature of both soy protein subunits. Aside from challenges associated with solubility, SPI dispersions can exhibit particularly high viscosity. The high viscosity of protein dispersions can often be a limiting factor during processing, as food systems with high viscosity are often responsible for fouling during heat treatment and particularly during water removal and concentration [24,25]. In addition, high viscosity protein dispersions can result in protein material remaining suspended in solution rather than becoming solubilised. This can result in inaccurate soy protein solubility values, which is the reason why a particular focus on soy protein solubility was undertaken in this study. In fact, Liu et al. (2011) showed that SPI dispersions at concentrations of 5%, w/w, protein displayed a weak-gel-like property during low oscillatory measurements [26]. These physicochemical properties make soy protein an ideal ingredient for some product applications such as plant-based yogurts, high protein powders, or soy-based beverages in the form of an oil-in-water emulsion; however, to the authors’ knowledge a commercially available soluble soy protein based beverage without oil addition does not exist. Therefore, based on the previous studies mentioned above, it is expected that the solubility may be improved and viscosity controlled through heat treating SPI dispersions above their denaturation temperatures under acidic or alkaline conditions prior to re- neutralisation, ultimately with the aim of creating a stable dispersion at neutral pH.

2. Results and Discussion 2.1. Influence of pH on ζ-Potential and Colour of Soy Protein Dispersions The ζ-potential values of dilute SPI dispersions measured as a function of pH ranged from 27.5 mV at pH 2.0 to −36.6 mV at pH 9.0 (Figure1). At pH values in the range 7.0 to 9.0, the ζ-potential did not change, while a significant decrease in the net negative charge was evident at pH values < 6.0. A cross-over at the isoelectric point occurred at ≈pH 4.2 (i.e., 0 mV) while at pH 4.0, the ζ-potential was +6.0 mV. However, it was not possible to measure ζ-potential at pH values close to the isoelectric point, due to protein precipitation. Then, the net positive charge of SPI dispersions continued to significantly increase up to 24.8 mV at pH 3 and only increased slightly further at pH 2.0 (27.5 mV). Malhotra and Coupland (2004) showed a similar trend for ζ-potential of SPI dispersions as a function of pH, with the ζ-potential reaching 0 mV between pH 4 and 5 [14] and Molecules 2021, 26, x FOR PEER REVIEW 3 of 16

Molecules 2021, 26, 3015 24.8 mV at pH 3 and only increased slightly further at pH 2.0 (27.5 mV). Malhotra3 and of 15 Coupland (2004) showed a similar trend for ζ-potential of SPI dispersions as a function of pH, with the ζ-potential reaching 0 mV between pH 4 and 5 [14] and with approximate valueswith approximate of +20 and −50 values mV at of pH +20 2 andand −8,50 respectively. mV at pH 2Probably and 8, respectively. the most important Probably point the frommost Figure important 1 is that point there from was Figure no significant1 is that thereincrease was in nothe signiﬁcant net negative increase charge infrom the ≈ netpH 7.0negative to 9.0, charge while fromit is ≈wellpH understood 7.0 to 9.0, while [22] itthat is well an understoodincrease in [pH22] results that an increasein substantial in pH improvementsresults in substantial in solubility, improvements which is in often solubility, attributed which tois an often increase attributed in electrostatic to an increase repul- in sion.electrostatic repulsion.

0 2345678910 -10 -potential (mV) -potential

ζ -20

-30

-40

-50 pH

Figure 1. Zeta potential as a function of pH for soy protein isolate dispersions. Figure 1. Zeta potential as a function of pH for soy protein isolate dispersions. The effect of pH on the colour profile of SPI dispersions is shown in Table1. Increasing the pHThe from effect pH of 6.9 pH (control on the colour pH) to profile pH 9.0 resultedof SPI dispersions in decreases is shown in the values in Table for 1.L *Increas- (63.8 to ing43.5), thea *(pH− 2.13from to pH−3.19) 6.9 (control and b* (7.12 pH) toto 1.63)pH 9.0 parameters, resulted in which decreases is indicative in the values of a decrease for L* (63.8in whiteness to 43.5), anda* (−2.13 an increase to −3.19) in and yellow b* (7.12 and to blue 1.63) hue. parameters, The changes which in theis indicativeL* valuewere of a decreasepartly reversible, in whiteness as shown and an in increase Table1, oncein yellow the pH and was blue adjusted hue. The back changes to pH in 6.9; the however, L* value werea* and partlyb* values reversible, decreased as shown further. in This Table seems 1, once to be the in pH general was agreementadjusted back with to the pH study 6.9; however,of Jiang et a* al. and (2010), b* values who decreased found that further. adjusting This the seems pH of to SPI be in to general pH 12, equilibratingagreement with for the1 h, study before of re-adjusting Jiang et al. (2010), back to who pH 7.0found resulted that adjusting in a lower the turbidity pH of SPI than to SPI pH dispersions 12, equili- bratingprepared for at 1 pHh, before 7.0 [21 re-adjusting]. However, back decreasing to pH 7.0 the resulted pH from in 6.9 a lower to 2.0 turbidity did not result than inSPI a dispersionssignificant change prepared in L at* (63.8 pH 7.0 to 62.6)[21]. orHowever,a* values decreasing (2.13 to −2.62) the pH with from only 6.9 a minor to 2.0 decreasedid not * resultin the inb avalues significant (7.12 change to 5.57). in This L* (63.8 is indicative to 62.6) or of a* the values SPI dispersion(2.13 to −2.62) changing with only to a a slightly minor decreasedarker, green in the and b* values blue dispersion.(7.12 to 5.57). After This readjustment is indicative ofof pHthe backSPI dispersion to pH 6.9, changing the L* value to * * areduced slightly furtherdarker, togreen 57.91, and with bluea dispersion.and b values After returning readjustment to their of originalpH back values. to pH 6.9, Again, the Lthis* value seems reduced to align further with data to 57.91, shown with by a Jiang* and etb* al.values (2010) returning when adjusting to their theoriginal pH of values. SPI to Again,pH 1.5 this before seems re-neutralising to align with to data pH 7,shown resulting by Jiang in very et littleal. (2010) change when in turbidityadjusting between the pH ◦ ofit SPI and to SPI pH dispersions 1.5 before re-neutralising at pH 7.0, when to pH measured 7, resulting at 20 in C[very21 little]. It change is well in understood turbidity betweenthat adjusting it and theSPI pHdispersions of SPI dispersions at pH 7.0, when to extremely measured acidic at 20 or °C alkaline [21]. It is environments well under- stoodcauses that a significant adjusting change the pH in of the SPI tertiary dispersion and quaternarys to extremely structure acidic of or both alkalineβ-conglycinin environ- mentsand glycinin, causes resultinga significant in protein change dissociation in the tertiary and and the formationquaternary of structure subunits, of which both will β- conglycininultimately affect and glycinin, colour properties. resulting in protein dissociation and the formation of subunits, which will ultimately affect colour properties. Table 1 also shows the effect of pH on the colour profile of heat-treated SPI dispersions. Comparing the colour of dispersions heat-treated at pH 6.9 to the dispersion at pH

Molecules 2021, 26, 3015 4 of 15

Table 1. Color chromaticity coordinates of unheated and heated (90 ◦C × 20 min) soy protein isolate dispersions at pH 2.0, pH 6.9, and pH 9.0 and dispersions readjusted from pH 9.0 and pH 2.0 back to pH 6.9.

pH 2.0 pH 6.9 pH 9.0 pH 6.9 Adjusted after pH 2.0 pH 6.9 Adjusted after pH 9.0 Unheated L* 62.6 ± 0.60 a 63.8 ± 0.67 a 43.5± 0.04 c 57.9 ± 4.26 b 59.2 ± 0.47 b a* −2.62 ± 0.07 b −2.13 ± 0.07 b −3.19 ± 0.03 c −1.92 ± 0.19 a −4.07 ± 0.0 d b* 5.57 ± 0.07 b 7.12 ± 0.22 a 1.63 ± 0.03 c 7.36 ± 0.19 a 0.08 ± 0.08 d Heated L* 59.6 ± 0.83 b 63.8 ± 2.58 a 61.2 ± 0.58 b 60.6 ± 0.94 b 67.4 ± 0.55 a a* −2.37 ± 0.19 b −2.89 ± 0.29 b −3.09 ± 0.03 c −0.97 ± 0.21 a −2.03 ± 0.12 b b* 2.55 ± 0.27 d 5.46 ± 0.07 c 5.41 ± 0.03 c 8.58 ± 0.38 a 7.27 ± 0.32 b Values are the means of data ± standard deviations with values within a row not sharing a common superscript differing signiﬁcantly (p < 0.05). L*-value denotes white-black, a*-value denotes green-red, b*-value denotes yellow-blue.

Table1 also shows the effect of pH on the colour proﬁle of heat-treated SPI dispersions. Comparing the colour of dispersions heat-treated at pH 6.9 to the dispersion at pH 9.0 showed a decrease in the L* (63.8 to 61.19) and a* values (−2.89 to −3.09) with the b* value remaining unchanged (5.46 to 5.41). This was visually represented as a slightly darker dispersion; however, the changes were not as large as their unheated counterparts. Meanwhile, the SPI dispersion heat-treated at pH 2.0 resulted in a decrease of the L* (63.8 to 59.6) and b* values (5.46 to 2.55), and an increase in the a* value (−2.89 to −2.37) compared to the heated dispersion at pH 6.9. This resulted in a darker dispersion with an increase in the blueness corresponding to the decrease in the b* value. After readjustment of the SPI dispersions to the original pH 6.9, the post pH 2 dispersions showed a darker, greener, and yellower appearance, as shown by the decrease in the L* value (63.8 to 60.6) and an increase both a* (−2.89 to −0.97) and b* (5.46 to 8.58) values. The heat- treated pH 9.0 dispersion showed a lighter, redder, and yellower appearance, which was represented as an increase in L* (63.8 to 67.38), a* (−2.89 to −2.03), and b* values (5.46 to 7.27). Light microscopy images (Figure2) of SPI dispersions measured at pH 2.0, 6.9, and 9.0 before and after heat treatment at 90 ◦C × 20 min show that heat-treated samples have a less deﬁned particle boundary and appear to be more homogenously distributed throughout the solution. However, particulate material is still clearly visible in all heat- treated dispersions (Figure2D–F).

2.2. Particle Size and Protein Solubility Analysis of Soy Protein Dispersions Particle size distribution profiles of unheated and heated (90 ◦C × 20 min) SPI dispersions are shown in Figure3. The size of the particles in unheated SPI dispersions ranged from 10 to 600 µm, with all dispersions displaying monomodal distributions (Figure3A), similar to a number of previous studies [27,28]. Unheated and heated SPI dispersions at pH 6.9 had weighted mean values (D4,3) of 130 and 101 µm, respectively, with unheated and heated SPI dispersions at pH 2.0 having significantly (p < 0.05) smaller D4,3 values of 57.2 and 52.1 µm, respectively. The mean particle size was significantly (p < 0.05) smaller in the heated SPI dispersions at pH 9.0 (D4,3 = 54.6 µm) than the respective unheated samples (D4,3 = 128 µm), with a bimodal size distribution (Figure3B). The particle size data presented in the current study are comparable to those of other plant protein dispersions, such as those from pea and fava bean protein [29,30]. The reduction in particle size in heated dispersions at pH 2.0 and 9.0 indicate that some protein dissociation did occur but certainly not complete break-down into individual subunits. Peng et al. (2020) found a similar particle size distribution profile for unheated soy protein dispersions measured across a range of pH values [22]. Molecules 20212021,, 2626,, 3015x FOR PEER REVIEW 55 ofof 1516

Molecules 2021, 26, x FOR PEER REVIEW 6 of 16 Figure 2.2. LightLight microscopymicroscopy imagesimages of of soy soy protein protein isolate isolate dispersions dispersions before before heat heat treatment treatment at pHat pH 2.0 2.0 (A), (A pH), pH 6.9 (6.9B), ( andB), and pH pH 9.0 (C) and after heat treatment at 90 °C for 20 min at pH 2.0 (D), pH 6.9 (E), and pH 9.0 (F). 9.0 (C) and after heat treatment at 90 ◦C for 20 min at pH 2.0 (D), pH 6.9 (E), and pH 9.0 (F). 2.2. Particle Size and Protein Solubility Analysis of Soy Protein Dispersions Particle size distribution profiles of unheated and heated (90 °C × 20 min) SPI dispersions are shown in Figure 3. The size of the particles in unheated SPI dispersions ranged from 10 to 600 μm, with all dispersions displaying monomodal distributions (Figure 3A), similar to a number of previous studies [27,28]. Unheated and heated SPI dispersions at pH 6.9 had weighted mean values (D4,3) of 130 and 101 μm, respectively, with unheated and heated SPI dispersions at pH 2.0 having significantly (p < 0.05) smaller D4,3 values of 57.2 and 52.1 μm, respectively. The mean particle size was significantly (p < 0.05) smaller in the heated SPI dispersions at pH 9.0 (D4,3 = 54.6 μm) than the respective unheated samples (D4,3 = 128 μm), with a bimodal size distribution (Figure 3B). The particle size data presented in the current study are comparable to those of other plant protein dispersions, such as those from pea and fava bean protein [29,30]. The reduction in particle size in heated dispersions at pH 2.0 and 9.0 indicate that some protein dissociation did occur but certainly not complete break-down into individual subunits. Peng et al. (2020) found a similar particle size distribution profile for unheated soy protein dispersions measured across a range of pH values [22].

FigureFigure 3. 3. ParticleParticle size size of of soy soy protein protein isolate isolate dispersions dispersions without without (A)( Aand) and with with heat heat treatment treatment (B) (B) ◦ (90(90 °CC ×× 2020 min) min) at at pH pH 6.9 6.9 (―― (——), ),pH pH 9.0 9.0 (─ (– ─ – ─ –)) and pH 2.0 (····).(‧‧‧‧).

ToTo further further investigate investigate the the effect effect of ofheating heating on onthe the functional functional properties properties of SPI of SPIdisper- dis- sionspersions and andextrapolate extrapolate the reduction the reduction in particle in particle size to sizesolubility, to solubility, dispersions dispersions were centri- were fuged at 800 g and the solids content suspended in the supernatant was measured. The solubility of SPI dispersions was significantly influenced by changes in both pH and thermal treatment (Figure 4). The solubility of unheated SPI dispersions at pH 9.0 (28.8%) was significantly higher than in dispersions at pH 6.9 (17.8%). However, when the pH was reduced from 9.0 to 6.9, a slight decrease in solubility was observed (i.e., 26.4%; Figure 4). The solubility of unheated SPI dispersions at pH 2.0 was 36.2%, which was higher than the 21% solubility when the pH was readjusted back to pH 6.9. It has previously been reported [30,31] that the solubility of SPI can be displayed as a U-shaped curve at which the lowest point is close to the isoelectric point between pH 4.0 and 5.0, with near 100% solubility achieved at both pH 2.0 and pH 9.0. However, the solubility values shown in the present study at pH 2.0 and 9.0 are lower than the values presented in the aforementioned studies. This may be due to the differences in the SPI production processes, whereby in the present study, a commercially produced SPI was used, as opposed to native soy protein. Heat treatment was shown to increase the solubility of all samples compared to their respective unheated samples (Figure 4), with solubility values of 24.8, 72.8, and 69.0% for heated dispersions at pH 6.9, 9.0, and 2.0. This may be contrary to some studies where the thermally induced denaturation/aggregation may be associated with a reduction in solubility; however, as far back as Kinsella (1979) [32], who stated that while it is commonly acknowledged that low heat treatment equates to better solubility, it may not always be the case. This in part is due to changes in the protein structure, particularly the glycinin fraction, at temperatures greater than the denaturation temperature. However, what seems to be more significant is the pH at which heat treatment is performed. Heat-treating soy protein in a semi-dissociated state at extremes of pH alters the structure of the protein,

Molecules 2021, 26, x FOR PEER REVIEW 7 of 16

Jiang et al. (2010), who reported that the dissociation of the SPI complexes at 21 °C can Molecules 2021, 26, 3015 lead to the exposure of hydrophobic groups previously occluded in the native agglomer-6 of 15 ates [21]. They also reported a restructuring of protein secondary structural characteristics in the 11S and the 7S subunits after exposure to extreme pH conditions (pH 1.5 or pH 12) for 1 h at room temperature before pH readjustment back to pH 7. Interestingly, the ζ- centrifuged at 800 g and the solids content suspended in the supernatant was measured. potential remained relatively constant when changing the pH from 6.9 to 9.0, as shown in The solubility of SPI dispersions was significantly influenced by changes in both pH and Figurethermal 1, indicating treatment that (Figure electrostatic4). The solubility repulsion of between unheated amino SPI dispersionsacid subunits at pHis obviously 9.0 (28.8%) notwas the significantlyonly contributory higher factor than infor dispersions the increased at pH solubility 6.9 (17.8%). under However, severe alkaline when the condi- pH was tions.reduced Readjusting from 9.0 the to pH 6.9, from a slight 9.0 and decrease 2.0 to in 6.9 solubility after heat was treatment observed resulted (i.e., 26.4%; in solubility Figure4 ). valuesThe of solubility 36.6 and of 47.8%, unheated respectively, SPI dispersions which is at a pH decrease 2.0 was in 36.2%, solubility which compared was higher to the than solubilitythe 21% at solubility pH 2.0 and when 9.0, but the it pH may was be readjustedattributed to back the topartial pH 6.9. re-folding It has previouslyof the protein been subunits.reported Another [30,31] point that theto mention solubility is ofthat SPI th cane addition be displayed of NaOH as a and U-shaped HCl during curve pH at whichad- justmentthe lowest may pointincrease isclose the ionic to the strength isoelectric or Na pointCl content between of the pH dispersions. 4.0 and 5.0, However, with near the 100% amountssolubility added achieved would at seem both to pH contribute 2.0 and pH very 9.0. little However, effect theon the solubility actual valuesSPI solubility, shown in as the Jiangpresent et al. study(2010) at[21] pH found 2.0 and that 9.0 only are lowerNaCl concentrations than the values ≥ presented100 mM, substantially in the aforementioned higher thanstudies. in the Thispresent may study, be due had to thea significant differences effect in the on SPI SPI production solubility, processes, which was whereby attributed in the to apresent decrease study, in electrostatic a commercially repulsion. produced SPI was used, as opposed to native soy protein.

100 90 80 70 60 50 40 Solubility (%) Solubility 30 20 10 0 Unheated Heated

Figure 4. Solubility of unheated and heated (90 ◦C × 20 min) soy protein isolate dispersions measured Figure 4. Solubility of unheated and heated (90 °C × 20 min) soy protein isolate dispersions meas- at pH 6.9 ( ), pH 9.0 ( ), pH 2.0 ( ), and pH 6.9 readjusted after heat treatment at pH 9.0 ( ) and ured at pH 6.9 (□), pH 9.0 (■), pH 2.0 (■), and pH 6.9 readjusted after heat treatment at pH 9.0 (■) andpH pH 6.9 6.9 readjusted readjusted after after heat heat treatment treatment at at pH pH 2.0 2.0 ( (■).). Heat treatment was shown to increase the solubility of all samples compared to their 2.3. Influence of pH and Heat Treatment on Viscosity of Soy Protein Dispersions respective unheated samples (Figure4), with solubility values of 24.8, 72.8, and 69.0% for heatedViscosity dispersions profiles for at pHSPI 6.9,dispersions 9.0, and at 2.0. pH This 2.0, may6.9, and be contrary9.0 before to and some after studies heat treat- where mentthe (90 thermally °C × 20 inducedmin) are denaturation/aggregationshown in Figure 5. The heat may treatment be associated of 90 °C with for a20 reduction min was in selectedsolubility; as the however, denaturation as far backtemperature as Kinsella for (1979)both major [32], who components stated that of whileSPI occurs it is commonly below or atacknowledged ≈90 °C [22,33]. that Note low that heat while treatment the exact equates production to better conditions solubility, of the it may commercial not always SPI be (heatthe treatment, case. This pH-induced in part is due precipitation, to changes in etc.) the are protein unknown, structure, the thermal particularly treatment the glycinin ap- pliedfraction, in this at study temperatures was deemed greater sufficient than the to denaturation exceed denaturation/aggregation temperature. However, conditions. what seems A sodiumto be more dodecylsulphate signiﬁcant is thepolyacrylamide pH at which gel heat electrophoresis treatment is performed. (SDS-PAGE) Heat-treating profile of the soy SPIprotein indicates in asome semi-dissociated initial protein state denaturation, at extremes as ofshown pH alters by a thedecrease structure in protein of the protein,band intensityallowing obtained the solvent under access non-reducing to the newly conditions exposed (Appendix pockets ofA.2: secondary Figure A1; structures lane 1) com- within paredthe protein,to those aidingobserved hydration in the reducing and solubilisation. lane (Appendix These resultsA.2: Figure are also A1; in lane agreement 2). For all with unheatedJiang et and al. (2010),heated who SPI dispersions, reported that the the shear dissociation stress increased of the SPI with complexes increasing at 21 shear◦C can rate lead (Figureto the 5), exposure indicating of hydrophobicthat the dispersions groups previouslywere non-Newtonian occluded in shear-thinning the native agglomerates fluids (Fig- [21]. ureThey 5). Table also 2 reported data show a restructuring that the flow ofbehaviour protein secondaryindex (n) was structural initially characteristics 0.86 for unheated in the SPI11S dispersions and the 7S at subunitspH 6.9 and after was exposure lower for to extremeSPI dispersions pH conditions at pH 2 (pH(n = 1.50.76) or and pH pH 12) for9 (n 1 h = 0.43).at room However, temperature after heat before treatment pH readjustment (90 °C × 20 backmin), to the pH n 7.values Interestingly, decreased the inζ SPI-potential dis- persionsremained at pH relatively 6 and pH constant 2.0, but when they changing remained the constant pH from at 6.9pH to 9.0. 9.0, This as shown decrease in Figurein n 1, indicating that electrostatic repulsion between amino acid subunits is obviously not the only contributory factor for the increased solubility under severe alkaline conditions. Readjusting the pH from 9.0 and 2.0 to 6.9 after heat treatment resulted in solubility values of 36.6 and 47.8%, respectively, which is a decrease in solubility compared to the solubility Molecules 2021, 26, 3015 7 of 15

at pH 2.0 and 9.0, but it may be attributed to the partial re-folding of the protein subunits. Another point to mention is that the addition of NaOH and HCl during pH adjustment may increase the ionic strength or NaCl content of the dispersions. However, the amounts added would seem to contribute very little effect on the actual SPI solubility, as Jiang et al. (2010) [21] found that only NaCl concentrations ≥100 mM, substantially higher than in the present study, had a signiﬁcant effect on SPI solubility, which was attributed to a decrease in electrostatic repulsion.

2.3. Influence of pH and Heat Treatment on Viscosity of Soy Protein Dispersions Viscosity profiles for SPI dispersions at pH 2.0, 6.9, and 9.0 before and after heat treatment (90 ◦C × 20 min) are shown in Figure5. The heat treatment of 90 ◦C for 20 min was selected as the denaturation temperature for both major components of SPI occurs below or at ≈90 ◦C[22,33]. Note that while the exact production conditions of the commercial SPI (heat treatment, pH-induced precipitation, etc.) are unknown, the thermal treatment applied in this study was deemed sufficient to exceed denaturation/aggregation conditions. A sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) profile of the SPI indicates some initial protein denaturation, as shown by a decrease in protein band intensity obtained under non-reducing conditions (Appendix A.2: Figure A1; lane 1) compared to those observed in the reducing lane (Appendix A.2: Figure A1; lane 2). For all unheated and heated SPI dispersions, the shear stress increased with increasing shear rate (Figure5), indicating that the dispersions were non-Newtonian shear-thinning fluids (Figure5). Table2 data show that the flow behaviour index ( n) was initially 0.86 for unheated SPI dispersions at pH 6.9 and was lower for SPI dispersions at pH 2 (n = 0.76) and pH 9 (n = 0.43). However, after heat treatment (90 ◦C × 20 min), the n values decreased in SPI dispersions at pH 6 and pH 2.0, but they remained constant at pH 9.0. This decrease in n value indicates an increase in shear-thinning behaviour. The consistency coefficient (K) values of SPI dispersions at pH 6.9 and pH 2.0 were 0.14 and 0.36 Pa.s, respectively, whereas at pH 9.0, the K value was significantly higher at 6.99 Pa.s, indicating a higher overall viscosity. Conversely, for SPI dispersions measured after the heat treatment, an inverse in K values was observed, with samples at pH 6.9, 2.0, and 9.0 having K values of 6.97, 7.90, and 1.42 Pa.s, respectively.

Table 2. Rheological parameters, consistency coefﬁcient, and ﬂow behavior index of soy protein isolate dispersions (10%, w/w, protein) as a function of pH before and after heat treatment.

pH Consistency Coefﬁcient Flow Behavior Index K (Pa.s) n pH 6.9 0.14 ± 0.01 e 0.86 ± 0.02 b pH 2.0 0.36 ± 0.00 d 0.76 ± 0.03 c pH 9.0 6.99 ± 0.01 b 0.43 ± 0.02 d pH 6.9 * 6.97 ± 0.07 b 0.33 ± 0.02 e pH 2.0 * 7.90 ± 0.04 a 0.36 ± 0.03 e pH 9.0 * 1.42 ± 0.00 c 0.48 ± 0.02 d pH 6.9 after heating @ pH 9.0 0.16 ± 0.00 e 0.74 ± 0.01 c pH 6.9 after heating @ pH 2.0 0.02 ± 0.00 f 0.90 ± 0.03 a * Samples heated at 90 ◦C × 20 min. Values are the means of data ± standard deviations; a–f values within a column not sharing a common superscript differ signiﬁcantly (p < 0.05). Molecules 2021, 26, x FOR PEER REVIEW 8 of 16

value indicates an increase in shear-thinning behaviour. The consistency coefficient (K) values of SPI dispersions at pH 6.9 and pH 2.0 were 0.14 and 0.36 Pa.s, respectively, whereas at pH 9.0, the K value was significantly higher at 6.99 Pa.s, indicating a higher overall viscosity. Conversely, for SPI dispersions measured after the heat treatment, an Molecules 2021, 26, 3015 inverse in K values was observed, with samples at pH 6.9, 2.0, and 9.0 having K values 8of of 15 6.97, 7.90, and 1.42 Pa.s, respectively.

Shear stress (Pa) 30

0 0 50 100 150 200 250 300 350 Shear rate (s-1)

Figure 5. Flow curves of unheated (Black) and heat-treated (90 ◦C × 20 min) (Grey) soy protein Figureisolate 5. Flow dispersions curves of (10%, unheatedw/w) at(Black) pH 2.0 and (---- heat-tre; ----ated), (90 pH °C 6.9 × (20——— min) (Grey);——— soy) andprotein pH 9.0 (—; isolate dispersions (10%, w/w) at pH 2.0 (- - - -; - - - -), pH 6.9 (–– –– ––; –– –– ––) and pH 9.0 (―; ―), —), and soy protein isolate dispersions measured at pH 6.9 after heat treatment at pH 2 (—) and and soy protein isolate dispersions measured at pH 6.9 after heat treatment at pH 2 (—) and pH 9 pH 9 (—). (—). At pH 6.9, the shear stress of unheated SPI dispersions increased from 0.8 to 18.5 TablePa 2. over Rheological a shear parameters, rate ramp consistency of 9.6 to 300 coefficient, s−1, while and adjustingflow behavior the index pH fromof soy 6.9protein to 2.0 re- isolatesulted dispersions in an increase (10%, w/w in, protein) shear stress as a function from 1.5 of topH 27.9 before Pa and (Figure after5 heat). The treatment. most significant difference (p < 0.05) amongst the unheated samples was the SPI dispersion adjusted to pH Consistency Coefficient Flow Behavior Index pH 9.0, which showed a significantly higher viscosity than dispersions at pH 2.0 and pH 6.9 K (Pa.s) n (Figure5 ). Initially, the shear stress was 18.2 Pa at a shear rate of 9.6 s−1 and increased pH 6.9 0.14 ± 0.01 e 0.86 ± 0.02 b up to 78.5 Pa at 300 s−1. This may be due to the dissociation of β-conglycinin (7S) and pH 2.0 0.36 ± 0.00 d 0.76 ± 0.03 c glycinin (11S) into subunits and an increase in protein polarity at the high pH, resulting in pH 9.0 6.99 ± 0.01 b 0.43 ± 0.02 d a concomitantpH 6.9 increase * in water-binding capacity.6.97 ± 0.07 Previously,b a study 0.33 showed ± 0.02 e an increase ≈ in water-holdingpH 2.0 * capacity in commercial 7.90 soy ± 0.04 protein a from 2 up to 0.36 10 ±g 0.03 of water/ge of protein whenpH increasing9.0 * the pH from 3.5 to 1.42 7.5 ± [0.0022]. c Heat-treated SPI 0.48dispersions ± 0.02 d at pH 6.9 andpH pH6.9 after 2.0 had heating a significantly @ pH 9.0 higher viscosity 0.16 ± 0.00 compared e to unheated 0.74 samples, ± 0.01 c which was attributedpH 6.9 after to heating the additional @ pH 2.0 denaturation/aggregation 0.02 ± 0.00 f of the two major 0.90 ± protein 0.03 a fractions ◦ * Samplesin SPI, heatedβ-conglycinin at 90 °C × and20 min. glycinin, Values havingare the means denaturation of data ± temperaturesstandard deviations; of ≈72 a–fvalues and 90 C, withinrespectively a column not [6]. sharing However, a commo heatingn superscript SPI at pH differ 9.0 wassignificantly the only (p sample < 0.05). to result in a lower viscosity compared to its unheated counterpart (Figure5 and Table2). Native glycinin is a hexamerAt pH 6.9, [34] the comprised shear stress of six of subunits unheated with SPI eachdispersions subunit increased made up offrom an 0.8 acidic to 18.5 and Pa basic overpeptide a shear (covalently rate ramp linkedof 9.6 to via 300 a disulphide s−1, while adjusting bond); as the a result, pH from it is the6.9 non-covalentto 2.0 resulted bonds in an (hydrophobicincrease in shear and stress electrostatic from 1.5 interactions) to 27.9 Pa (Figure between 5). The the subunits,most significant which difference account for (p the < 0.05)differences amongst in the solubility unheated and samples viscosity was across the theSPI pH dispersion scale [35 adjusted,36]. Under to extremepH 9.0, which acidic or showedalkaline a significantly conditions, higher the non-covalent viscosity than linkages dispersions weaken, at andpH 2.0 dissociation and pH 6.9 of (Figure the subunits 5). can occur. This dissociation can result in the exposure of previously buried hydrophobic residues, and in conjunction with high negative electrostatic repulsion, it can cause high viscosity in SPI dispersions. However, after heating to 90 ◦C at pH 9.0, the exposed hydrophobic residues may result in the re-arrangement of the subunits into a more soluble aggregated form, causing a significant decrease in viscosity. Similarly, β-conglycinin comprised of three subunits, α, α0, and β, linked via non-covalent interactions can disso- Molecules 2021, 26, 3015 9 of 15

ciate as the electrostatic repulsion between them increases at pH values away from the isoelectric point.

2.4. Temperature Related Viscoelastic Properties of Soy Protein Dispersions To investigate the heating and cooling effect on the visco-elastic properties of SPI dispersions, low-amplitude oscillatory measurements were carried out as a function of temperature (Figure6). SPI dispersions at pH 2.0 showed more elastic than viscous properties as the temperature increased above 65 ◦C. The G’ increased rapidly reaching a maximum of 62.7 Pa at 80 ◦C. Thereafter, the G0 decreased sharply to 29.7 Pa as the temperature was reduced from 80 to 40 ◦C. A similar trend was measured for the G”, as temperature increased, so too did the viscous moduli, peaking at 13.6 Pa, before reducing when the temperature peaked at 80 ◦C. As temperature started to decrease, G” increased before peaking at 17.4 Pa at 73 ◦C. The data are indicative of the formation of a weak gel within the SPI dispersion (Note: all SPI dispersions displayed flow properties under high shear after heat treatment). The results of the current study are in broad agreement with those of previous studies [29,37], in which weak gel-like structures were observed in pea and soy protein emulsions, respectively. SPI dispersions at pH 6.9 showed contrasting behaviour, in which G0 was initially lower than G” until the temperature peaked at 80 ◦C, after which G0 increased sharply, peaking at 32.8 Pa at 40 ◦C. G” followed a similar trend, and it remained lower than the G0, increasing to only 7.64 Pa. Conversely, the SPI dispersion at pH 9.0 showed a profile in which the G0 was initially higher than the G” value, at 147 and 23.9 Pa, respectively. This may be due to the dissociation of soy protein into subunits, causing an increase in the critical protein volume fraction. However, both G0 and G” moduli decreased as temperature increased, with G0 decreasing to 12.7 Pa at 70 ◦C, before increasing to 18.6 Pa upon cooling to 40 ◦C. SPI dispersions at pH 9.0 displayed rheological behaviour consistent with a weak gel as G0 remained greater than G” throughout the temperature ramp. An explanation for the decrease in viscosity of the pH 9 dispersion may be due to the substantial changes in protein structure and exposure of hydrophobic groups after extreme alkaline treatment [38]. Frequency sweep profiles of SPI dispersions at pH 2.0, 6.9, and 9.0 are shown in Figure7 . All SPI dispersions initially showed G0 greater than G”, until approximately 10 Hz, indicating that up until this point, a weak gel network existed. Thereafter, the G” > G0 for all dispersions, signifying the transition from a weak gel to a more viscous liquid-like system [39]. Previously, Liu et al. (2011) showed that the critical concentration of an SPI dispersion to form a weak gel-like structure was approximately 5% (w/w, protein), with protein concentrations less than 5% (w/w) showing only viscous behaviour (i.e., G” > G0)[26]. Ultimately, the data in Figure7 confirm that the SPI dispersions have elastic-like behaviour at low frequencies but convert to viscous type systems at frequencies of ≈10 Hz or higher. This may have important knock-on effects in terms of beverage stability, particularly around hindering protein sedimentation during storage. Previously, Malhotra and Coupland (2004) showed good correlation between increased solubility with increasing viscosity in 5%, w/w, SPI dispersions [14]. Molecules 2021, 26, 3015 10 of 15 Molecules 2021, 26, x FOR PEER REVIEW 10 of 16

(A)

(B) (C)

Figure 6. Elastic ( G′0;; ------) )and and viscous viscous (G (G″”;; ─–– ─) modulus of soy protein isolate dispersions (10%, w/w) measured at pH 2.0 (A), pH 6.9 ( B) and pH 9.0 (C), as a function of temperature ((—―).).

Frequency sweep profiles of SPI dispersions at pH 2.0, 6.9, and 9.0 are shown in Fig- ure 7. All SPI dispersions initially showed G′ greater than G″, until approximately 10 Hz, indicating that up until this point, a weak gel network existed. Thereafter, the G″ > G′ for all dispersions, signifying the transition from a weak gel to a more viscous liquid-like system [39]. Previously, Liu et al. (2011) showed that the critical concentration of an SPI dispersion to form a weak gel-like structure was approximately 5% (w/w, protein), with protein concentrations less than 5% (w/w) showing only viscous behaviour (i.e., G″ > G′) [26]. Ultimately, the data in Figure 7 confirm that the SPI dispersions have elastic-like behaviour at low frequencies but convert to viscous type systems at frequencies of ≈10 Hz or higher. This may have important knock-on effects in terms of beverage stability, particularly around hindering protein sedimentation during storage. Previously, Malhotra and Coupland (2004) showed good correlation between increased solubility with increasing viscosity in 5%, w/w, SPI dispersions [14].

Molecules 2021, 26, x 3015 FOR PEER REVIEW 11 of 16 15

B C

Figure 7. Elastic (G0; - - -) and viscous (G”; ––) moduli of soy protein isolate dispersions at pH 2.0 (A), pH 6.9 (B), and pH ◦ Figure9.0 (C), 7. measured Elastic (G as′; ----) a function and viscous of frequency (G″; ─ ─ (Hz)) moduli at 40 ofC. soy protein isolate dispersions at pH 2.0 (A), pH 6.9 (B), and pH 9.0 (C), measured as a function3. Materials of frequency and (Hz) Methods at 40 °C. 3.1. Materials 3. Materials and Methods Non-hydrolysed soy protein isolate (SPI) powder was obtained from a commercial 3.1. Materials supplier. The total protein content, measured using Kjeldahl analysis (IDF 20-3:2004) of the SPINon-hydrolysed powder was 88.0%, soy proteinw/w, using isolate a conversion(SPI) powder factor was ofobtained 6.25. Non-protein from a commercial nitrogen supplier.content, measuredThe total protein according content, to the measured IDF standard using method Kjeldahl (Kjeldahl analysis analysis(IDF 20-3:2004) 20-4:2001) of thewas SPI 0.26%, powderw/w .was Moisture 88.0%, andw/w fat, using content a conversion was 4.39 andfactor 4.51%, of 6.25.w/w Non-protein, respectively, nitrogen and it content,contained measured calcium according (0.18%, w/w to), the sodium IDF standard (1.1%, w/w method), magnesium (Kjeldahl (0.04%, analysisw/w 20-4:2001)), potas- wassium 0.26%, (0.15%, w/ww/w. Moisture), chloride and (0.29%, fat contentw/w), and was phosphorous 4.39 and 4.51%, (0.75%, w/ww/w, respectively,), as obtained and from it containedthe commercial calcium supplier. (0.18%, The w/w pH), sodium of rehydrated (1.1%, w/w SPI), (5%, magnesiumw/w) was (0.04%, pH 6.9 w/w and), was potassium consid- (0.15%,ered as w/w the), control chloride pH (0.29%, for all w/w other), and analysis. phosphorous An SDS-PAGE (0.75%, proteinw/w), as proﬁle obtained ofthe from SPI the is commercialdescribed and supplier. shown The in Appendix pH of rehydratedA. SPI (5%, w/w) was pH 6.9 and was considered as the control pH for all other analysis. An SDS-PAGE protein profile of the SPI is de- scribed3.2. Zeta and Potential shown of in Soy Appendix Protein Dispersions A. Soy protein dispersions were prepared by adding SPI powder to deionised (DI) water 3.2.at 5%, Zetaw/w Potential, total of solids Soy Protein (20 ◦C). Dispersions The suspension was agitated using a magnetic stirrer (DynalonSoy protein Stir Bar dispersions Kit, VWR, Blanchardstown,were prepared by Dublin, adding Ireland) SPI powder at 350 to rpmdeionised for 1 h. (DI) After water 1 h atof 5%, stirring, w/w, total these solids dispersions (20 °C). were The suspension centrifuged wa ats 800 agitated× g for using 10 min, a magnetic and the stirrer supernatant (Dyn- alonwas removed.Stir Bar Kit, The VWR, initial Blanchardstown, pH of the dispersion Dublin, was Ireland) pH 6.9. at 350 Then, rpm the for pH 1 h. was After adjusted 1 h of stirring,using HCl these or NaOH,dispersions ranging were from centrifuged pH 2.0 toat 9.0.800× Then, g for the10 min, solids and content the supernatant was diluted was to removed.a ﬁnal concentration The initial pH of 0.002%, of the dispersionw/w, using wa DIs waterpH 6.9. and Then, an Eppendorf the pH was 5417R adjusted centrifuge using HCl(Eppendorf or NaOH, AG, ranging 22331 Hamburg,from pH 2.0 Germany). to 9.0. Then, Samples the solids were content measured was usingdiluted a Zetasizerto a final concentrationNano ZS (Malvern of 0.002%, Instruments w/w, using Ltd., DI Worcestershire, water and an Eppendorf UK). All measurements 5417R centrifuge were (Eppen- carried ◦ dorfout atAG, 25 22331C, in Hamburg, triplicate, inGermany). 10 mm polystyrene Samples were cuvettes measured using using a refractive a Zetasizer index Nano of 1.45. ZS

Molecules 2021, 26, 3015 12 of 15

3.3. Light Microscopy of Soy Protein Dispersions Soy protein dispersions were prepared by adding SPI powder to deionised (DI) water at 5%, w/w, total solids (20 ◦C) as described in Section 3.2 above but without the centrifugation step. The pH of SPI dispersions was adjusted from pH 6.9 using ≈3.5 mL of 1 M HCl or 1 M NaOH to pH 2.0 and 9.0, respectively. Then, sub-aliquots of samples were heat treated at 90 ◦C for 20 min in 50 mL Falcon tubes (Merck, Wicklow, Ireland) using a controlled- temperature water bath (GFL 1003, 30927 Burgwedel, Germany). The microstructure of unheated and heated samples was examined using an Olympus BX51 light microscope (Olympus BX-51, Olympus Corporation, Tokyo, Japan) under a 10× dry objective lens using differential interference contrast (DIC) at 20 ◦C. Images were taken using a Jenoptik C14 Imagic camera, and captured and analysed using Image Access Premium® 7 software (Wiltshire, UK).

3.4. Colour Analysis of Soy Protein Dispersions The colour of each dispersion (i.e., as prepared in Section 3.3) was expressed as L*, a*, and b* values using a Minolta Chroma Meter CR-400 colorimeter (Minolta Ltd., Milton Keynes, UK). SPI dispersions were prepared (5%, w/w, total solids), and their respective pH values were adjusted using NaOH and HCl accordingly. The dispersions were mixed at 20 ◦C for 15 min before measurements were performed. The L* value indicates lightness, a* values indicate redness–greenness, and b* values indicate yellowness–blueness. Analysis was performed in triplicate.

3.5. Particle Size and Solubility of Soy Protein Dispersions Soy protein dispersions (5%, w/w, total solids) were prepared as described in Section 3.3 above. The solubility analysis of SPI dispersions was performed by placing aliquots in 50 mL centrifuge tubes and centrifuging in a Thermo Scientiﬁc™, HeraeusTM Multifuge X1R Centrifuge (Thermo Scientiﬁc™, Waltham, MA, USA) at 800 g × 5 min at 25 ◦C. Solubility was expressed as the total solids content of the supernatant expressed as a percentage of the total solids content of the samples prior to centrifugation using a moisture analyser (CEM Smart System5™, Matthews, NC, USA). Measurements were performed in triplicate. Particle size distribution analysis of SPI dispersions was performed in triplicate using a laser light diffraction unit (Mastersizer, Malvern Instruments Ltd., Worcestershire, UK), equipped with a 300 RF lens. Particle and dispersant (i.e., water) refractive indices were set at 1.48 and 1.33, respectively. Measurements were recorded at laser obscuration levels >4% and at ~20 ◦C.

3.6. Viscosity and Low-Amplitude Oscillatory Measurements of Soy Protein Dispersions Soy protein dispersions (10%, w/w, total solids) were prepared by adding SPI powder to DI water (20 ◦C). The pH of SPI dispersions were adjusted from pH 6.9 using 1 M HCl or 1 M NaOH to pH 2.0 and 9.0, respectively. Then, sub-aliquots of samples contained in 50 mL conical Falcon tubes (Fisher Scientiﬁc, Bishop Meadow Road, Loughborough, Leicestershire, UK) were heat treated at 90 ◦C for 20 min by placing samples in a temperature-controlled water bath (GFL 1003, 30927 Burgwedel, Germany). Viscosity measurements of dispersions were performed at 25 ◦C using a controlled stress rheometer (TA Instruments, Crawley, UK), equipped with a concentric cylinder geometry performed at a shear rate ranging from 9.6 to 300 s−1 over 3 min. Viscosity measurements were performed on samples before and after heat treatment. The power law applied to the log–log plots of shear stress (τ) versus shear rate (R) was used to analyse non-Newtonian ﬂuids as described by:

τ = KRn Molecules 2021, 26, 3015 13 of 15

where τ is the shear stress (Pa), R is the shear rate (s−1), K is the consistency coefficient (Pa.sn), and n is the flow behaviour index [34]. Values of n = 1, n < 1, and n > 1 indicate Newtonian, shear thinning, and shear thickening flow behaviour, respectively [35]. Low-amplitude oscillatory measurements of unheated SPI dispersions (10%, w/w) were analysed using the same rheometer and geometry as above at pH values of 2.0, 6.9, and 9.0. The temperature was increased from 40 to 80 ◦C at a gradient of 2.5 ◦C min−1, held at 80 ◦C × 3 min, and decreased to 40 ◦C at 2.5 ◦C min−1. Oscillatory measurements were recorded at a frequency of 1 Hz and 0.5% strain, giving both the storage (G0) and loss (G00) moduli. Subsequently, a mechanical spectrum was performed at a frequency ranging from 0.1 to 100 Hz (frequency dependence of elastic, G0, and viscous, G”, moduli) at 25 ◦C (0.5% strain). Frequency sweeps were determined in triplicate within the linear viscoelastic region to ensure non-destructive rheological analysis.

3.7. Statistical Analysis Analysis of variance (ANOVA) was carried out using the Minitab 17 (Minitab Ltd., Coventry, UK, 2007) statistical analysis package, and the effects of treatment and replicates were estimated for response variables. Tukey’s one-way multiple comparison test was used as a guide for pair comparisons of the treatment means. The level of signiﬁcance was determined at p < 0.05.

4. Conclusions This work has demonstrated that the solubility of SPI dispersions can be modified significantly using a combination of pH adjustment and heat treatment. Adjusting the pH of SPI dispersions to acidic (pH 2.0) or alkaline (pH 9.0) conditions increased solubility, with heat treatment at these pH values working synergistically to increase solubility even further. The use of acidic/basic pH conditions influences the hydroelectric charge on the soy proteins causing subunit dissociation, and in doing so facilitates alternative protein–protein interactions which would otherwise not be possible. This study has shown that the relatively simple adjustment of pH prior to thermal heat treatment followed by pH readjustment can result in a more soluble protein dispersion at neutral pH values, with pH 2 treated dispersions showing a greater solubility after heat treatment and re- neutralisation. Knowledge of these underlying properties, which are responsible for SPI dispersion behaviour, allow for the manipulation of protein functionality in beverage products (i.e., reduced viscosity and greater solubility), and should provide a platform for further work in this area. Particularly, as SPI is often subjected to multiple heating steps during the production of nutritional formulations, the optimal stage in the process for heat treatment at acidic/alkali conditions may yet need to be elucidated.

Author Contributions: Conceptualisation, N.A.M., J.A.O. and S.A.H.; data curation, T.D.O. and N.A.M.; formal analysis, T.D.O. and N.A.M.; funding acquisition, D.F.M.D. and N.A.M.; methodology, N.A.M.; project administration, J.A.O. and N.A.M.; resources, S.A.H., D.F.M.D. and N.A.M.; supervi- sion, J.A.O. and N.A.M.; writing—original draft, T.D.O.; writing—review and editing, T.D.O., J.A.O., and S.A.H., N.A.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Abbott Nutrition and the Teagasc Agriculture and Food Development Authority Walsh Scholarship scheme under project MDDT0565. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Appendix A Appendix A.1. Sodium Dodecylsulphate Polyacrylamide Gel Electrophoresis The protein proﬁle of the SPI powder was determined using sodium dodecylsulphate- polyacrylamide gel electrophoresis (SDS-PAGE). The powder sample was dissolved under Molecules 2021, 26, x FOR PEER REVIEW 14 of 16

Appendix A Molecules 2021 26 , , 3015 Appendix A.1. Sodium Dodecylsulphate Polyacrylamide Gel Electrophoresis 14 of 15 The protein profile of the SPI powder was determined using sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The powder sample was dis- solvedreducing under and reducing non-reducing and non-reducing conditions in conditions an SDS buffer; in an 10 SDSµL buffer; of each 10 buffer μL of was each added buffer to waswells added in a 12% to wells Bis-Tris in Nu-PAGEa 12% Bis-Tris gel and Nu-PAG electrophoreticE gel and analysiselectrophoretic was performed analysis using was anper- X- formedCell Surelock using an electrophoresis X-Cell Surelock unit electrophoresi (Novex Technologies,s unit (Novex Fischer Technologies, Scientiﬁc, Blanchardstown, Fischer Scien- tific,Dublin, Blanchardstown, Ireland) at a constant Dublin, voltageIreland) of at 180 a constant V for 55 voltage min. Samples of 180 forV for SDS-PAGE 55 min. Samples analysis forcontained SDS-PAGE 1 µg analysis protein contained per µL of sample1 μg protein buffer per solution. μL of sample Gels were buffer then solution. stained Gels overnight were thenusing stained 0.05% overnight (w/v) Coomassie using 0.05% brilliant (w/v blue) Coomassie R-250 in brilliant 25% (v/v blue) isopropanol R-250 in 25% and ( 10%v/v) (iso-v/v) propanolacetic acid. and After 10% staining, (v/v) acetic the gelsacid. were After de-stained staining, the using gels a 10%were (v/v de-stained) isopropanol using and a 10% 10% ((v/vv/v)) isopropanol acetic acid solution and 10% until (v/v) a acetic clear acid background solution wasuntil achieved. a clear background was achieved.

AppendixAppendix A.2. A.2. Supplementary Figure

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