Kottahachchi Kankanamge, Manjula Nishanthi (2018) Physico-chemical and functional properties of whey proteins as affected by the source of whey. PhD thesis, Victoria University.

Abstract

Increasing global demand for fermented dairy products and salted hard cheese varieties has led to the vast generation of acid and salty whey waste streams. Unlike sweet whey, acid and salty whey are not conveniently processed to whey protein concentrates (WPC) or isolates (WPI) due to compositional variations mainly characterized by elevated lactic acid and salt concentrations, respectively. Therefore, the necessity of developing a suitable processing method in converting acid and salty whey waste streams to usable concentrate powders is importantly emphasized by the dairy industry. Factors affecting the behavior of the whey proteins (WPs) present in these two whey streams, during or post-production are unknown. Hence, the present study was proposed to establish the physico-chemical behavior of the WPs present in acid and salty whey and their behavior during application of traditional concentration and spray drying. To gain an insight into the applicability of these WPC powders in commercial applications as food ingredients, current study further proposed evaluating their molecular, physical and functional property changes during storage. The proposed outcomes were achieved in five separate phases. During the first phase, the physico-chemical characteristics of WPs in sweet, salty and acid whey were investigated and compared with those of native whey, which underwent minimum commercial processing. Acid whey used in the current study was generated during Greek yoghurt manufacturing. Four whey streams were analyzed for the composition, interactional properties and molecular structures. WPs from acid whey were characterized by hydrophobically and covalently driven protein aggregation. Covalent aggregation in acid whey consisted of both thiol/disulphide and non-thiol/disulphide mediated reactions. Fourier transform infrared (FTIR) data characterized this protein aggregation as β-sheet attractions causing subtle changes in the secondary structure. In contrast, WPs in salty whey aggregated via van der Waals, hydrogen, electrostatic interactions and covalent bonds. Both thiol/disulphide and non-thiol/disulphide interactions led to cross-linked β-sheets, disrupting the secondary protein structures. This aggregation exposed hydrophobic segments while oxidizing a high number of free thiol groups. The absence of these types of WP aggregation in sweet or native whey highlighted the fact that elevated salt concentration in salty whey or heat treatment applied during production of acid whey are largely responsible for structural differences. In second phase, the influence of physico-chemical characteristics of liquid-WPCs obtained by ultrafiltration of acid and salty whey streams on the surface composition, particle organization, secondary structures and protein interactions of the respective spray dried WPC powders was investigated. Their properties were compared with those of native and sweet whey. Acid whey concentrate demonstrated characteristically low surface charge, high surface hydrophobicity, high average particle size and high thiol activity compared with sweet and native whey concentrates. Salty whey concentrate was characterized by low surface hydrophobicity, high thiol activity and low average particle size. Surface characterization of WPC powders revealed protein-rich surfaces for all whey powders while those in salty whey were highly hydrophobic. Protein characteristics of native and sweet whey WPC powders largely followed those of concentrates. In contrast, protein characteristics of the acid and salty whey WPC powders largely changed from those of the liquid WPCs. In third phase, changes of the secondary structure and protein interactions of WPs present in native, sweet, acid and salty-WPC powders were analyzed following storage at 4, 25 or 45 °C and 22 or 33% relative humidity (RH) for a period of 90-days. WPs aggregated predominantly through covalent crosslinking, achieving maximum at 45°C and 33% RH. Greater participation of β-lactoglobulin (β-LG) in covalent crosslinking was evident in all WPC powders, while that of α-lactalbumin (α-LA) was significantly (p < 0.05) high in acid- WPC powder only. Reaction order of β-LG denaturation in acid and salty-WPCs was approximately 2, while approximately 1 in native and sweet-WPC powders. Activation energy was significantly (p < 0.05) higher in native and sweet-WPC powders, with averages recorded as 97 and 49.8 kJmol-1, respectively, than that in acid and salty-WPC powders with averages of 27.5 and 33.8 kJmol-1, respectively, mainly attributed to the inherently high concentrations of lactic acid (LA) and salts in these WPC powders. Storage conditions may compromise stability of WPs in dry state, which is also influenced by their inherent composition. Thus, in fourth phase, physical characteristics of native, sweet, acid and salty-WPC powders were analyzed during storage at several temperatures (4, 25, 45°C) and RH levels of 22 and 33% for a period of 90 days. Particle surface of native, sweet and acid-WPC powders was dominated by proteins under all storage conditions, while fat and minerals prevailed on the surface of salty-WPC powder. Compared to native- and sweet-WPC powders, origin of acid- and salty-WPC powders influenced these streams to be rich in minerals, primarily accumulated in the particle core. Hydrophilic nature of the core impacted redistribution of proteins within the particle during storage. Compared to native- and sweet-WPC powders, particles were cohesively arranged in acid- and salty- WPC powders, which in turn changed physical properties such as particle size and surface charge. Elevated storage temperatures induced protein denaturation, melting of surface free fat and lactose crystallization in WPC powders, while humidity regulated the molecular mobility in these reactions. In addition to the influence on physical characteristics, combined impact of storage conditions and composition of WPC powders may affect the functional characteristics of WPs thus affect their functionality. Therefore, in the fifth phase, functional characteristics of native, sweet, acid and salty-WPC powders were analyzed after storage at 25°C and RH levels of 22 and 33% for 90 days. Native-, sweet- and acid-WPC powders exhibited a high solubility (97–82%), which was largely retained during storage. In contrast, the solubility of salty-WPC started at ~52-55% and gradually increased by ~5% during storage. Ionic sodium in salty-WPC interlinked WPs through salt bridges and charge screening, exposing reactive sites for intensive aggregation. Heat stability of salty-WPC was the highest (64s), while lowest was recorded for native-WPC (16s). In the presence of ionic sodium in salty-WPC, WPs denature due to ionic-bridging, charge screening and osmotic effects leading to intensive aggregation. High emulsion activity was recorded for salty-WPC powder, while those for other WPC powders were similar. Emulsion stability varied as native-> acid-> salty- > sweet-WPC. High number of hydrophobic segments was likely exposed on the protein surface in salty-WPC powders due to sodium-induced WP denaturation, thus increasing the number of proteins absorbed to the emulsion interface, enhancing the emulsion activity. Functionality of different WPC powders predominantly depended on the inherited composition and storage conditions. In conclusion, the inherent composition of acid and salty whey streams affects physico-chemical properties of WPs present in these whey streams, their liquid WPCs and spray dried WPC powders. Furthermore, properties of WPs present in these WPC powders were influenced by the compositional differences, processing effects of these two whey streams and storage conditions, which can be observed at molecular level, in turn affecting their physical characteristics. Furthermore, compositional differences showed a combinational impact with storage conditions on the functional properties of acid and salty WPC powders. Therefore, the unique composition of acid and salty whey is a factor that must be manipulated strategically in order to achieve a commercially viable production process and storable WPC powders.

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