TY - BOOK
T1 - Characterisation of Controlled Aggregation of α-lactalbumin
T2 - Modulation by Heat, Calcium and Cysteine concentration
AU - Nielsen, Line Ravn
PY - 2018
Y1 - 2018
N2 - Whey, a by-product from cheese production, is a premium raw material to a wide range of protein ingredients. Over the years, costumer demands has increased calling for enhanced documentation of the high nutritional quality of whey proteins and their functional properties. This has created an interest and a need of the development of more sophisticated and tailored ingredients, that challenge the original processing steps in the production of the protein ingredients. This can lead to undesired changes such as both physical and chemical changes of the proteins. However, not all changes are undesired and enabling the controlled formation of physical changes could be also advantageous for the industry in a business perspective. An example of an undesired change that could also be a potential desired physical change is aggregation of α-lactalbumin (α-LA), a protein considered not to easily aggregate due to the absence of a free thiol group in the protein structure. α-LA is the second most abundant protein in whey and a key ingredient in infant formulas due to a favourable nutritional profile high in the amino acid tryptophan. In the present study, the aim was to obtain controlled thermal aggregation at reduced heat loads to enhance future development of whey protein ingredients. A systematic study of the effect of heating temperature, calcium concentration and the addition of a small molecular mass thiol, cysteine on unfolding and aggregation of α-LA was performed. The aggregates were characterised by the use of complementary analytical methods including differential scanning calorimetry, intrinsic fluorescence spectroscopy, size exclusion chromatography, multi angle light scattering and gel electrophoresis. The results showed that marked aggregation was obtained when α-LA was heat treated at high temperature (90 °C) and aggregation was initiated by thermal cleavage of disulphide bonds resulting in aggregates comprising unfolded α-LA species. The addition of cysteine concentrations of 0.01-0.1 molar ratios (0.14-1.4 mM) to disulphide bond equivalents in α-LA promoted the formation of soluble aggregates. This effectively decreased the holding time required to reach a particular amount of aggregates with a defined size. Excess cysteine (>1.4 mM) caused a destabilisation of α-LA, shown by decreased denaturation temperature and at an even higher cysteine concentration (>14 mM) gel formation was observed.Furthermore, cysteine promoted the aggregation of α-LA at lower heating temperatures (25, 50, and 70 °C), which resulted in a reduced heat load required to obtain aggregation. In addition, cysteine addition allowed aggregation to occur even at temperatures below the denaturation temperature of the calcium saturated (~68 °C) and the calcium depleted (~37 °C) forms of α-LA. Analysis of aggregation kinetics showed that the addition of cysteine increased the rate of aggregation. In addition, α-LA was found to form compact spherical aggregates with low value intrinsic viscosities. The ability to tune aggregate size and compactness ranging from small aggregates to large compact aggregates and gels demonstrated in the present investigation opens up for further studies and use of α-LA aggregates in food applications ranging from high-protein beverages where aggregates contribute little to viscosity to the use as structuring agents in high-viscosity or gelled food products.
AB - Whey, a by-product from cheese production, is a premium raw material to a wide range of protein ingredients. Over the years, costumer demands has increased calling for enhanced documentation of the high nutritional quality of whey proteins and their functional properties. This has created an interest and a need of the development of more sophisticated and tailored ingredients, that challenge the original processing steps in the production of the protein ingredients. This can lead to undesired changes such as both physical and chemical changes of the proteins. However, not all changes are undesired and enabling the controlled formation of physical changes could be also advantageous for the industry in a business perspective. An example of an undesired change that could also be a potential desired physical change is aggregation of α-lactalbumin (α-LA), a protein considered not to easily aggregate due to the absence of a free thiol group in the protein structure. α-LA is the second most abundant protein in whey and a key ingredient in infant formulas due to a favourable nutritional profile high in the amino acid tryptophan. In the present study, the aim was to obtain controlled thermal aggregation at reduced heat loads to enhance future development of whey protein ingredients. A systematic study of the effect of heating temperature, calcium concentration and the addition of a small molecular mass thiol, cysteine on unfolding and aggregation of α-LA was performed. The aggregates were characterised by the use of complementary analytical methods including differential scanning calorimetry, intrinsic fluorescence spectroscopy, size exclusion chromatography, multi angle light scattering and gel electrophoresis. The results showed that marked aggregation was obtained when α-LA was heat treated at high temperature (90 °C) and aggregation was initiated by thermal cleavage of disulphide bonds resulting in aggregates comprising unfolded α-LA species. The addition of cysteine concentrations of 0.01-0.1 molar ratios (0.14-1.4 mM) to disulphide bond equivalents in α-LA promoted the formation of soluble aggregates. This effectively decreased the holding time required to reach a particular amount of aggregates with a defined size. Excess cysteine (>1.4 mM) caused a destabilisation of α-LA, shown by decreased denaturation temperature and at an even higher cysteine concentration (>14 mM) gel formation was observed.Furthermore, cysteine promoted the aggregation of α-LA at lower heating temperatures (25, 50, and 70 °C), which resulted in a reduced heat load required to obtain aggregation. In addition, cysteine addition allowed aggregation to occur even at temperatures below the denaturation temperature of the calcium saturated (~68 °C) and the calcium depleted (~37 °C) forms of α-LA. Analysis of aggregation kinetics showed that the addition of cysteine increased the rate of aggregation. In addition, α-LA was found to form compact spherical aggregates with low value intrinsic viscosities. The ability to tune aggregate size and compactness ranging from small aggregates to large compact aggregates and gels demonstrated in the present investigation opens up for further studies and use of α-LA aggregates in food applications ranging from high-protein beverages where aggregates contribute little to viscosity to the use as structuring agents in high-viscosity or gelled food products.
UR - https://soeg.kb.dk/permalink/45KBDK_KGL/fbp0ps/alma99122110664005763
M3 - Ph.D. thesis
BT - Characterisation of Controlled Aggregation of α-lactalbumin
PB - Department of Food Science, Faculty of Science, University of Copenhagen
ER -