Primary and double emulsification properties of sonicated milk systems with different casein to whey protein ratios
Creators: name: Silva, Mayumi
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 300602
subject: Food chemistry and food sensory science
classificationCode: 400405
subject: Food engineering

description: Emulsions are increasingly being utilized in the food industry as delivery systems for functional ingredients. As emulsions are thermodynamically unstable systems, they tend to release the entrapped materials over time under various environmental stress conditions. The stability or the encapsulation efficiency of the emulsions are directly dependent on the emulsification technology and the composition of the aqueous phase, oil phase and the emulsifiers/surfactants. Thus, the combination of efficient emulsification technologies and appropriate ingredient formulations will contribute to obtain emulsions with greater stability, encapsulation protection and a targeted control release. Therefore, this study aimed to develop milk protein stabilized primary and double emulsions consisting with varying casein to whey protein ratios with the use of low frequency ultrasound.
 
In the first stage of this study, the effect of sonication on the physicochemical and structural characteristics of milk protein solutions with varying  casein:whey (C:W) ratios of 80:20, 60:40, 50:50 and 40:60 using milk protein isolate (MPI) and whey protein isolate (WPI) as casein and whey protein sources was studied. Low frequency ultrasound at 20 kHz was applied under different energy densities corresponding to the sonication times of 0-10 min. The results showed that sonication affected the physicochemical and structural characteristics of milk proteins depending on the C:W ratio and the sonication time. Ultrasound induced the dissociation, denaturation and unfolding of ß-lactoglobulin into random coil structures, which thereby led to aggregation through ß-sheet intermolecular crosslinking. These aggregates had different bonding mechanisms and individual protein involvement where; milk protein solutions with a higher portion of caseins produced more hydrophobically driven aggregates while whey-rich milk protein solutions produced more disulphide mediated aggregates.
 
In the second stage of the study, interfacial properties of the sonicated milk protein solutions and ultrasound-assisted emulsification properties of milk protein solutions were studied. Grape seed oil (GSO) was used as the oil phase as it has better nutritional, physicochemical and sensory properties. Emulsions were prepared under different sonication times of 1-10 min. As a consequence of ultrasound-induced protein structural changes, sonicated milk protein solutions showed differences in the dynamic interfacial tension at the GSO-water interface compared to the native solutions. Emulsification properties were primarily governed by the C:W ratio in the continuous phase and the sonication time. Increase in sonication time reduced the particle size while the minimum treatment time required to produce stable emulsions was reduced with increasing whey protein fraction. The composition and structure of the interfacial layer of emulsions also depended on the C:W ratio.
 
Based on the interfacial and emulsification properties of the primary emulsions, the most efficient sonication times (7 and 10 min) fit for all milk protein ratios were selected. The physical and oxidative stability of the emulsions prepared under 7 and 10 min sonication were analysed for 10 days of period at 4 °C of storage temperature as the third stage of the study. Results revealed that the physical and chemical stability of primary emulsions is dependent on C:W composition and sonication time. Sono-emulsification for 10 min exhibited storage instability depending on the C:W ratio where whey-rich emulsions were more susceptible for oxidation while casein-rich emulsions showed the physical instability through a depletion flocculation mechanism. In terms of both physical and oxidative stabilities, 7 min sonicated emulsions showed better storage stability for 10 days period when the aqueous phase contained an optimum milk protein concentration of ≥40% whey proteins.

In the fourth stage of the study, the in-vitro digestion behaviours of 7 min sonicated emulsions were determined. It was found that the in-vitro digestion behaviour of emulsions was influenced by the C:W ratios and the ultrasound-induced structural changes and modification of bonding behaviours of proteins at the interface. Within the gastric phase, higher pepsin digestion rate was observed in C:W ratio of 60:40, 50:50, 40:60 and MPI. WPI emulsions increased the resistant to pepsin digestion and emulsions remained more gastric stable. The intestine phase was influenced by the microstructural properties of the stomach digesta where digestion rate was reduced in casein-rich emulsions as more flocculated and coalesced droplets were entered into the intestine phase. The free fatty acid release rate followed the order for emulsions stabilized with; 40:60&gt; WPI&gt; MPI&gt; 50:50¿ 60:40. Therefore, under the tested conditions, WPI and C:W ratio of 40:60 can be considered as the best two compositions to stabilize primary emulsions using ultrasound-assisted emulsification.

Formation of double emulsion was the next challenging stage of the study. Water-oil-water type double emulsions were prepared using ultrasound-assisted two-step emulsification method. C:W ratio of 60:40, 50:50, 40:60, MPI and WPI were used as internal and external aqueous phases and GSO as the oil phase. Emulsification and stability properties of primary and double emulsions were analysed and the in-vitro digestion behaviour of the double emulsions was also determined as a preliminary investigation. The size of the secondary droplets was dependent on the C:W ratio although, the size of the secondary droplets were not affected by the size of the primary emulsion droplet. The highest encapsulation efficiency was observed in WPI and C:W ratio of 40:60 implying the whey-rich emulsions were more efficient in the encapsulation process. Emulsions with higher initial droplet size and/or encapsulation efficiency were more unstable during storage due to the release of entrapped molecules and water from the inner aqueous phase. The in-vitro digestion of milk protein stabilized double emulsions were also influenced by the C:W ratio in the aqueous phases. Whey-rich double emulsions were more successful in the protection of entrapped materials during the stomach phase allowing release of oil at the intestinal phase.
 
Therefore, milk protein stabilized primary and double emulsions can be prepared under sono-emulsification with careful control of composition of the aqueous phase, oil phase and emulsifiers under optimum ultrasound conditions. Under the optimum sonication conditions, C:W ratio of 40:60 and WPI can be considered as the most suitable milk protein compositions to develop primary and double emulsions. Therefore, this study provides important suggestions that can be used to improve the encapsulation efficiency, stability and the control release of oil in dairy based emulsion systems.