Effects of lyotropic series salts on the functional properties of bambara groundnut (Voandzeia subterranean) protein isolate

Lawal, O.S., Sodeinde, K.O., Adediran, A.A., Nishinari, K., Olatunji, O.S., Nelana, S.M. and Ayanda, O.S. Department of Industrial Chemistry, Federal University Oye-Ekiti, P.M.B 373, Oye-Ekiti, Ekiti State, Nigeria Department of Mechanical Engineering, Landmark University, Omu Aran, Kwara State, Nigeria. Glyn O. Phillips Hydrocolloid Research Centre, School of Food and Pharmaceutical Engineering, Faculty of Light Industry, Hubei University of Technology, Wuhan 430068, China School of Chemistry and Physics, University of KwaZulu-Natal, Westville, Durban 4000, South Africa Department of Chemistry, Vaal University of Technology, Vanderbijlpark 1900, South Africa


Introduction
Studies on the hydrodynamics of protein molecules in a three-component system like protein-water-salt have gained a lot of attention in recent years. Salt solutions have remarkable effects on the structure and functional properties of hydrocolloids (Yin et al., 2008). These effects are sensitive to the nature of the salt which may be chaotropic or kosmotropic. Kosmotropic salts stabilize the structure of proteins while chaotropic salts destabilize protein structures. These terms were later extended to the apparently correlating property of increasing or decreasing, respectively, the structuring of water. Chaotropes interrupt the hydrogen-bonded network of water, allowing macro-molecules more structural freedom and encouraging protein extension and denaturation. Kosmotropes are stabilizing solutes, which increase the order of water, whereas chaotropes create weaker hydrogen bonding, decreasing the order of water, increasing its surface tension and destabilizing macromolecular structures (Uedaira and Uedaira, 2001).
The stabilizing or destabilizing effects of salts on proteins generally follows the lyotropic or Hofmeister series which is a qualitative ordering of ions based originally on their propensity to salt out proteins from aqueous solutions (Kunz et al., 2004). Generally, the ranking of the ions in descending order of effectiveness in driving processes such as folding, subunit assembly, crystallization and precipitation of protein is as follows; anions: SO 4 2-> H 2 PO 4 -> CH 3 COO -> Cl -> Br -> I -> ClO 4 -> SCNand cations: NH 4 + > Cs + > K + > Na + > Li + > Mg 2+ > Ca 2+ > Ba 2+ The Hofmeister phenomenon is more pronounced for anions than cations. In homogeneous protein systems, the predominant attractive forces linking protein molecules are short-range hydrogen bonding, hydrophobic, electrostatic and van der Waals interactions (Halasz and Lasztity, 2017). Salts such as Na 2 SO 4 , NaCl, NaBr, NaI, NaClO 4 and NaSCN affect the physicochemical properties and interactions between proteins by their concentration and the ionic strength effects they have on charged groups of the protein molecules. Previous studies have shown that the two effects of salts make major contributions to the structure-stabilizing properties of the proteins, and these are their effects on solvent (water) structure and electrostatic interactions with the charged groups of the protein (Sarabia et al., 2000;Li et al., 2013). Ion effect on protein stability is assumed to be employed through two operative forces: (i) surface tension and (ii) preferential hydration. The net free energy of stabilization, δ(ΔGº) stability (Equation (1)) depends on the change in cavity formation, δ(ΔGº) cavitation (surface tension effect) and the free energy contribution from protein-solvent interaction, (δ(ΔGº) salvation (hydration).
The measure of the efficiency of different ions in affecting proteins in solutions depends on a combination of free energy of cavitation, which is positive (favourable) for a solvent with increased surface tension and free energy of solvation which depends upon the nature of the protein-solvent interaction (Breslow and Guo, 1990). Hofmeister salt effects on proteins were attributed to the favourability of cavity formation for a cosolute ion in the protein solvation shell. It was demonstrated that the surface tension increment σ of salt can give a quantitative amount of the measure of the salting-in and salting-out effects of electrolytes on proteins. The surface tension increment σ is defined from Equation (2): where γ is the solution surface tension and m is the salt concentration.
In addition, a previous study posited that the surface charge density of ions correlates well with the stability of nucleic acid triplexes and it was suggested that the same correlation would apply to protein stability. The position of ions in the Hofmeister ranking correlates with the Jones-Dole equation (Equation 3), which establishes a relationship between these salt-solvent effects and the viscosity of the salt solution (η) with salt concentration c, relative to the viscosity of water (η o ): The coefficient A is an indication of the strength of electrostatic interaction of the salt in solution while the B -viscosity coefficient indicates ion -solvent interactions. The term D is only needed at very high salt concentrations. A√c represents the electrostatic Debye -Hückel effects. Chaotropic ions have negative Bviscosity coefficients which make them deactivate proteins, while kosmotropic ions have positive Bviscosity coefficients and are expected to be stabilizers. In addition, preferential interactions of cosolutes with the protein surface have been suggested for the kosmotropic and chaotropic effects (Lin and Timasheff, 1996).
In previous work, the effects of anions on thermally induced whey protein isolate were reported and findings revealed that monovalent salts have a universal effect on the fracture properties of thermally induced whey protein isolate gels regardless of anion valence or Hofmeister effects (Bowland and Foegeding, 1995). The effect of the Hofmeister series on gluten aggregation and strength have been reported and the findings have shown that aggregation time followed the order of the Hofmeister series, with minor effects at salt concentration <0.3 M and increasing differences at higher salt concentrations (Melnyk et al., 2011). The effects of Hofmeister series salts on emulsions, foams and gels of jack bean protein has been reported and findings have revealed that functional properties of the protein isolate were significantly affected by the Hofmeister salts (Lawal, 2005). The functional properties of native and chemically modified Voandzeia subterranean concentrates have been reported and studies showed that functional properties such as foaming, emulsifying and gelation properties were dependent on the temperature, pH and ionic strength of the solutions (Lawal et al., 2007).
The applications of seed proteins in food systems or any other relevant application depend extensively on their functional properties which are controlled by the forces linking protein molecules. These forces influence functional properties such as solubility, foaming, emulsifying, and gelation capacity. Seed proteins are very important for diverse applications and the understanding of the forces in an aqueous solution that controls their physico-chemical processes is crucial in fields such as food processing, process design in the chemical industry, drug design in the pharmaceutical industry and the modelling of biological processes. Plant protein isolates have vital applications particularly as additives in food systems (Bainy et al., 2010). Isolation and characterization of some seed proteins from lesserknown sources have been reported in the literature. They include mucuna (Adebowale and Lawal, 2003;Lawal and Adebowale, 2004), African locust bean (Lawal, 2004;Lawal et al., 2005), Lablab bean (Lawal, 2005), broad bean (Arogundade et al., 2006), Jack bean (Lawal and Adebowale, 2006), bayberry , kidney bean (Yin et al., 2008), and field pea (Adebiyi and Aluko, 2011). In the present investigation, seed protein was isolated from Voandzeia subterranean also known as bambara groundnut. Voandzeia subterranean is an underutilized legume (Adebowale et al., 2002), it is widely cultivated throughout tropical Africa, India, Sri Lanka, Indonesia and Malaysia (Hillocks et al., 2011). Being underutilized, the functional properties of Voandzeia subterranean seed proteins are not well known. This study would provide an understanding of (1) (3) eISSN: 2550-2166 © 2022 The Authors. Published by Rynnye Lyan Resources FULL PAPER the functional properties of the Voandzeia subterranean seed protein under the influence of kosmotropic and chaotropic anions. This is the first report on the effect of cosolutes or additives in the aqueous colloidal system of Voandzeia subterranean seed protein and the findings are important for its applications.

Materials and reagents
Voandzeia subterranean seeds were obtained from Bodija Market, Ibadan, Nigeria. Na 2 SO 4 , NaCl, NaBr, NaI, NaClO 4 , NaSCN and NaOH, all analytical grades were purchased from Sigma-Aldrich (St. Louis, MO). The average yield of protein isolate from the flour was 28.4%, whereas, the percentage protein content of the protein isolate was 92.2%. These were determined using the Kjeldahl method. Crude nitrogen of the samples was determined in triplicate by the standard micro-Kjeldahl method then converted to protein content using a 6.25 conversion factor (AOAC, 1985).

Preparation of protein isolate
Protein isolates were prepared using the method previously described by Lawal et al. (2007). Alkaline solubilisation was used because the process facilitates higher yield based on preliminary studies. Voandzeia subterranean seeds were dehulled manually after soaking for 3 hrs. It was air-dried for 48 hrs at room temperature (30±2°C), and then dry-milled to fine powders before sieving using 75 μm sieve. Flour (1 kg) was dispersed in distilled water (10 L) and the pH was adjusted to 8.0 with 1 mol/L NaOH, to facilitate protein solubilisation. It was stirred at a stirring speed of 250 rpm for 4 hrs at 30±2 o C. The pH of the supernatant obtained after centrifuging at 4000×g (Type GLC-1 Ivan Sorvall Inc., USA) for 30 mins was adjusted from 8.0 to 4.0 with 0.5 mol/L HCl to precipitate the protein isolate, which was recovered by centrifugation at 5000 g for 30 mins. The protein isolates in cake form were dried with a freeze dryer.

Foaming capacity and foam stability
Foaming capacity and foam stability were studied using the method of Akintayo et al. (1999). The protein isolate (2 g) was dispersed in 100 mL of 0.1, 1.0 and 2.0 mol/L solutions of Na 2 SO 4 , NaCl, NaBr, NaI, NaClO 4 and NaSCN. The solutions were whipped vigorously for 2 min using Phillips kitchen blender. The volumes of the solutions were recorded before and after whipping. The percentage volume increase, which serves as the index of foam capacity, was calculated using Equation 4.
Where V 1 is the volume of solution before whipping; V 2 is the volume of protein solution after whipping. The volume of foam that remained after 8 hrs standing (30±2°C) was expressed as the percentage of the initial volume for the foam stability.

Emulsifying properties
Emulsifying properties were determined using the method of Neto et al. (2001). Protein solution (5 mL, 10% w/v) prepared in 0.1, 1.0 and 2.0 mol/L solutions of Na 2 SO 4 , NaCl, NaBr, NaI, NaClO 4 and NaSCN were homogenized with 5 mL of corn oil (Executive Chef Unilever, Lagos, Nigeria) for 1 min using Vari-Whirl mixer set at speed 2 (A901, Salver Chem, Chicago, IL, USA). Thereafter, the emulsions were centrifuged at 1100×g for 5 mins. The height of the emulsified layer and that of the total contents in the tube was measured. The emulsifying activity (EA) was calculated using Equation 5.
The emulsion stability (ES) was determined by heating the emulsion at 80°C for 30 mins before centrifuging at 1100×g for 5 mins and was calculated using Equation 6.

Gelation properties
Gelation properties were studied using the method of Akintayo et al. (1999). Protein sample suspensions of 2 -20% w/v were prepared in 0.1, 1.0 and 2.0 mol/L solutions of Na 2 SO 4 , NaCl, NaBr, NaI, NaClO 4 and NaSCN. A 10 mL volume of each of the prepared dispersion was transferred into a test tube, which was heated in a boiling water -bath for 1 hr, followed by rapid cooling in a bath of cold water. The test tubes were cooled further at 4°C for 2 hrs. The least gelation concentration was taken as the concentration when the sample from the inverted tube did not fall or slip.

Water holding capacity
The water holding capacity was determined using the method of Lawal (2004). Protein concentrate sample (1 g) was mixed with 10 mL of 0.1, 1.0 and 2.0 mol/L solutions of Na 2 SO 4 , NaCl, NaBr, NaI, NaClO 4 and NaSCN for 30 s. The samples were allowed to stand at room temperature (30±2°C) for 30 mins before centrifuging at 5000×g for 30 mins. The volume of the supernatant obtained after centrifugation was determined in a 10 mL graduated cylinder. Water holding capacity was calculated per gram of total protein.

Statistical analysis
Measurements were carried out in triplicates and the (4)

Foaming properties
Foaming capacities and foam stabilities of Voandzeia subterranean protein isolate studied in the presence of Hofmeister ions and at different concentrations are presented in Figures 1 and 2, respectively. The results indicated that both foaming capacity and foam stability reduced as the ionic strength of the solution increased and the reductions in the values of foaming capacity and foam stability were significant at all ionic strengths (p<0.05). The highest foaming capacity (86.08%) and foam stability (78.1%) were recorded in protein solutions prepared with NaSCN (0.1 mol/L). The chaotropic salts (NaI, NaClO 4 and NaSCN) significantly (p<0.05) increased the foaming capacity and foam stability of the protein isolates than the kosmotropic salts (Na 2 SO 4 , NaCl and NaBr). The results also show that the increase in foaming capacity and foam stability followed the ranking in the Hofmeister series in the order: Na 2 SO 4 <NaCl<NaBr<NaI<NaClO 4 <NaSCN. According to the Jones-Dole equation (Equation 3), salts with chaotropic properties have negative B-viscosity coefficients which make them deactivate proteins. It is reasonable that kosmotropes, with positive B-viscosity tend to improve the inter-and intramolecular structuremaking interactions in aqueous solutions of protein while chaotropes weaken such interactions. In this sense, weakening the binding forces among the protein molecules could lead to increased flexibility among the macromolecules, thereby facilitating various changes in their kinetic behaviour as well. This enhanced flexibility accounts for the better foamability observed in protein solutions prepared with chaotropic salts. It has been reported that chaotropic salts may accelerate reactions of proteins by loosening their structure (Der and Ramsden, 1998;Zhao, 2016).
The instability of foams in colloids interfaces may be due to either foam coalescence or disproportionation. Coalescence involves the irreversible combination of two or more foam bubbles when the interfacial films drain and the bubbles are eventually ruptured to form a bigger bubble. Foam disproportionation is the process that involves the diffusion of air molecules through the disperse phase between bubbles. As a result of the increased Laplace pressure of smaller bubbles, the diffusion flux generally leads to the shrinking of smaller bubbles and the growth of larger gas bubbles, thereby leading to the instability of foams (Dickinson, 1992;Zhu et al., 2018). Generally, the denaturant action of Hofmeister ions among the chaotropes is due to the salting-in of the peptide groups and the improved interactions with the unfolded form of the protein (Kang et al., 2020). This process facilitates protein solubility and increases the concentration of the protein at the airwater interface which improves foam stability. The results also indicate that both foaming capacity and stability reduced as the ionic strength of the solution increased which is consistent with a report on the foaming properties of broad bean protein isolate (Arogundade et al., 2006).

Emulsifying properties
Both EA (Figure 3) and ES (Figure 4) showed ion specificity and were better in protein solutions in chaotropic environments than in kosmotropic environments. Accordingly, both the EA and ES reduced significantly (p<0.05) with an increase in salt concentration. Similarly, the increase in EA and ES followed the Hofmeister trend: Na 2 SO 4 <NaCl<NaBr<NaI<NaClO 4 <NaSCN although the results obtained in Na 2 SO 4 and NaCl solutions were not significantly different at all ionic strengths for EA and ES (p<0.05). An emulsion is a heterogeneous system of at least two immiscible liquid phases, one of which is dispersed in the other, in the form of droplets (Sadoc and Rivier, 2013;Tadros, 2016). Proteins act as a surfactant in many food emulsions diffusing to and being adsorbed at the oil-water interface. The migration of the protein  (Kulmyrzaev and Schubert, 2004). Chaotropic anions improved emulsifying activities because, in water, they are usually associated with large positive entropies (Damodaran, 1990;Hu et al., 2014), leading to the de-structuring of water molecules. Consequently, water gets more disordered and lipophilic in the presence of these anions, thus improving water-oil emulsions. In addition, the processes of the protein unfolding caused by protein denaturation facilitated the hydrophobic hydration which enhanced emulsifying activity. Emulsion systems are usually stabilized through physical entrapment of fat globules within the protein matrices largely via protein-protein interactions followed by the formation of an interfacial protein film that surrounds and stabilizes fat globules (Barbut, 1995;Su et al., 2000). The processes which cause emulsion instability are creaming, flocculation, coalescence and oiling off (Benhura and Chidewe, 2004). Low protein concentration often causes limited protein availability at the oil-water interface thereby causing flocculation and instability of emulsions. The extent of flocculation is dependent on the structure of the adsorbed layer and the thermodynamic quality of the intervening solvent. If the stabilizing film at the oil-water interface ruptures, this leads to coalescence, making the oil droplets merge into larger spherical globules. In creaming, oil rises in an oil-in-water emulsion (O/W) and this occurs when the density of oil droplets is less than that of the continuous phase. In this sense, improved ES in chaotropic protein solutions is a result of increased protein solubility which led to a higher concentration of protein in the oil-water interface.

Gelation properties
The gelation properties of Voandzeia subterranean protein isolates under various lyotropic salt concentrations are presented in Figure 5. Using the least gelation concentration (LGC) as the index of gelation. At various salt concentrations, the lowest LGCs were observed in protein solutions prepared in chaotropic salts. Progressive and significant (p<0.05) increase in LGC were observed as the ionic strength of the solution increased, following the Hofmeister trend: Na 2 SO 4 > NaCl > NaBr > NaI > NaClO 4 > NaSCN. A gel is a continuous network of macroscopic dimensions immersed in a liquid medium exhibiting no steady-state flow. In gelation, a three-dimensional network is developed by progressive aggregation of macromolecules or particles through chemical bonds or physical interactions under suitable conditions. The following processes occur in the heat-induced gelation process: Protein unfolding, water binding, proteinprotein interactions and water immobilization. Low salt concentration (0.1 mol/L) enhanced gelation better than higher salt concentrations (1.0 and 2.0 mol/L). At low salt concentrations, charge screening of protein molecules occurs as a result of the presence of counter ions thereby leading to decreasing electrostatic free energy of the protein. These processes may increase the activity of the solvent and protein solubility. However, when the salt concentration is high, the abundance of the salt ions reduces the solvating power of the solvent and this may reduce the protein solubility. In this sense, the reduction in protein solubility at higher salt concentration is as a result of the neutralization of charges on the protein molecules by the ions, which lead to weaker gel formation. Reduction in gel firmness of whey protein when the NaCl content of the mixture was increased has been reported (Otte et al., 1999). Similarly, an increase in the least gelation concentration has been reported for pigeon pea after the ionic strength of the medium was increased from 0.5 mol/L to 1.0 mol/L (Akintayo et al., 1999). These findings are also consistent with previous reports on soy protein (Li et al., 2007;Li et al., 2009). The denaturant action of chaotropic anions is due to the fact that they salt-in the peptide group and consequently they interact much more strongly with the unfolded form of a protein than with its native form, thereby pulling the protein unfolding reaction. It is reasonable that the enhanced gelation in protein solutions prepared with chaotropic salts could be attributed to an increase in the protein unfolding which allowed more effective networking among the protein

Water holding capacity
The water holding capacities of Voandzeia subterranean protein isolates obtained at various ionic strengths of Hofmeister salts are presented in Figure 6. In the dispersions prepared in 0.1 mol/L salt solutions, the highest water holding capacity (9.7 g/g) was observed in the solution of Na 2 SO 4 while the least was recorded in NaSCN solution. Generally, water holding capacity reduced progressively and significantly (p<0.05) as the concentration of the ions increased. This observation was consistent for both the chaotropic and the kosmotropic salts.
The results also indicate that kosmotropic salts facilitated improved water holding capacities over the chaotropic salts and generally, the reduction in the water holding capacity followed the Hofmeister trend: Na 2 SO 4 > NaCl > NaBr > NaI > NaClO 4 > NaSCN. Kosmotropic salts facilitate water structure stabilization while the reverse is observed with chaotropic salts (Uedaira and Uedaira, 2001;Bye et al., 2014). It has also been reported that direct ion binding to the peptide backbone by kosmotropic ions may facilitate structural stabilization while the chaotropic ions cause destabilization (Sarabia et al., 2000). As indicated in Equation 2, an increase in surface tension (γ) is enhanced by an increase in salt concentration (m). It is reasonable that the reductions in water holding capacity could be due to an increase in surface tension as the salt concentration increased. This is consistent with a previous report on the increase in surface tension with salt concentration (Persson et al., 2003;Jawerth et al., 2018). In addition, as water is more structured in the presence of kosmotropes, this may act as a driving force for its absorption by the protein molecules. Chaotropes may be considered as "sticky" ions with weakly structured water surrounding them, driven to non-polar surface and this explains the weaker water holding observed with the chaotropes.

Conclusion
Studies on the effect of various lyotropic salts and their concentrations on the functional properties of protein isolates of an underutilized but rich protein resource of Voandzeia subterranean is presented. The studies provide information about the influences of the various kosmotropic and chaotropic anions on the protein colloids which may be relevant in diverse applications such as food and pharmaceutical industries. Protein colloids prepared in chaotropic salts solutions (NaI, NaClO 4 and NaSCN) exhibited better foaming, emulsifying and gelling properties than in kosmotropic salts solutions (Na 2 SO 4 , NaCl, NaBr). However, water holding capacities were higher in protein dispersions in kosmotropic salts solutions than in chaotropic salts solutions. Findings reveal that Voandzeia subterranean protein isolates may be formulated for specific enduses by adjusting its co-additives. Being an underutilized seed protein resource, that is readily available in developing countries, it is expected that the results presented here would facilitate its applications in several industries where protein colloids are relevant. The salts used in this study have been used to understand the chemistry of Voandzeia subterranean protein isolates in food systems. Therefore, it is appropriate that food safety and regulatory standards should be consulted for the use of chemicals in food systems.