Ageing induced solubility loss of milk protein concentrate powder: Effect of protein conformational modifications, enthalpy relaxation and interaction with water

Md. Haque (2011). Ageing induced solubility loss of milk protein concentrate powder: Effect of protein conformational modifications, enthalpy relaxation and interaction with water PhD Thesis, School of Agriculture and Food Sciences, The University of Queensland.

       
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Author Md. Haque
Thesis Title Ageing induced solubility loss of milk protein concentrate powder: Effect of protein conformational modifications, enthalpy relaxation and interaction with water
School, Centre or Institute School of Agriculture and Food Sciences
Institution The University of Queensland
Publication date 2011-11
Thesis type PhD Thesis
Supervisor Andrew K Whittaker
Michael J Gidley
Bhesh R Bhandari
Hilton C Deeth
Total pages 147
Total colour pages 14
Total black and white pages 133
Language eng
Subjects 07 Agricultural and Veterinary Sciences
Abstract/Summary Abstract: Milk protein concentrate (MPC) is a dairy powder with high protein content, up to 85%. MPC powders are used as a food ingredient in a variety of applications for their high nutritional value and favorable taste. Generally MPC powders are associated with a gradual loss of solubility upon storage. It is believed that protein conformational modifications and water-protein interactions are two major factors that induce instability of protein and eventually affect solubility. To test these hypotheses MPC powder was stored at different water activities (aw 0.0 – 0.85) and temperatures (25 °C and 45 °C) for up to 12 weeks. Samples were examined periodically to determine (i) solubility, (ii) change in protein conformation by FTIR (coupled with principal component analysis (PCA) and partial least squared (PLS) analysis) and (iii) water status (interaction of water with protein molecules/surface) by measuring the transverse relaxation time (T2) with 1H-NMR (Nuclear Magnetic Resonance). Solubility of MPC decreased significantly with ageing time and this process was enhanced by increasing aw and storage temperature. Minor changes in protein secondary structure were observed with FTIR which seem unlikely to be sufficient to explain the change in solubility. PCA analysis confirmed this result. PLS analysis revealed some correlation between solubility and FTIR spectral features. This result suggests that unfolding of protein could be the initiation step of protein denaturation. NMR-T2 data suggested the presence of three distinct populations of water molecules (differing in their interaction with and distance from protein surfaces) and the proton signal intensity varied with the moisture content of sample. Generally MPC with added mineral salts retained solubility upon ageing more than control powder without additive. Mineral-induced change in MPC could be related to protein conformational modifications and interactions with water; this needs to be explored further for a better understanding of the mechanisms of solubility loss of MPC. For this purpose MPC powder was enriched with NaCl, KCl and sodium citrate in 1% and 10% (w/w salts per 100 g of protein) concentrations. After 60 days of storage at 25°C and at aw 0.23, the mineral-enriched powders were tested by FT-IR, followed by multivariate analysis, and NMR relaxometry. The FT-IR study showed no specific pattern (with respect to salt used) of protein unfolding upon storage. Also no significant change was observed in water-protein interaction patterns between control and salt-treated MPC. Higher solubility of mineral-enriched MPC compared to control was most probably due to changes to the physical structure of casein micelles and the balance between the hydrophobic interactions and electrostatic repulsions induced by interaction with mineral salt. Structural/enthalpy relaxation of protein molecules during storage may also have some effect on solubility of amorphous high-protein powders Characteristics of enthalpy relaxation under different storage conditions and its effect on solubility were investigated by Differential Scanning Calorimetry (DSC). A distinct pattern of enthalpy recovery of MPC powder was revealed by DSC, depending on aw, storage temperature and ageing time. The enthalpy value was much less for samples with lower moisture content (mc) (aw ≤ 0.23, mc. ≤ 5.5%) than for those with higher mc (aw ≥ 0.45, mc ≥ 8 %) for a particular storage period. This result suggests that the presence of more water molecules in close proximity to the protein surface facilitates kinetic unfreezing and subsequent motion of molecular segments of protein molecules towards thermodynamic equilibrium. The solubility of powder remained nearly unchanged upon enthalpy recovery. A similar time scale of enthalpy relaxation and solubility loss suggested that enthalpy relaxation within stored samples allows structural rearrangements that are responsible for subsequent solubility decreases. Changes in the structure and dynamics of protein segments could occur upon storage and, as a result of interaction with water and could have some link with solubility loss. Change in molecular mobility (or rigidity) in aged and fresh MPC was investigated by solid state 13C-NMR using cross polarization and direct polarization techniques. This study showed a minor change in mobility of molecular segments (backbone as well as side chain) upon storage of MPC at high aw for 14 weeks. This suggests that the rigidity of protein segments of non-aged MPC is slightly higher than that of aged MPC due to plasticization by water during storage at high aw, which could facilitate protein-protein interactions. Recent research suggests that solubility loss of MPC is related to changes in the surface of the powder particles due to interactions between casein micelles. Probing the changes occurring at different parts of powder particles spectroscopically may help to elucidate their molecular mechanisms. Differential changes in powder particles and similar changes in insoluble and soluble fraction of aged powder were examined by Raman and FT-IR techniques. No significant change in different parts of powder particles was observed by Raman microscopic analysis. The FT-IR study of isolated and freeze-dried insoluble and soluble fractions of MPC powder showed slightly higher unfolding of protein molecules of the aged, insoluble fraction of MPC powder compared to that of non-aged insoluble and non-aged soluble fractions. Combining all these study results, especially the findings of protein conformational changes (performed on aged, non-aged as well as soluble and insoluble fractions) and the similar time scale of enthalpy relaxation and solubility loss (from the thermal study), it could be suggested that there is a link between the limited protein unfolding/refolding and loss of solubility. Protein unfolding could be the initiation step of protein-protein interactions and subsequent changes in the microstructure of casein micelles at the surface of the powder particles that cause a decrease in solubility of MPC upon ageing.
Keyword Milk proteins
Solubility
Water Activity
Ft-IR
Principal Component Analysis
Proton NMR
13C NMR
Enthalpy Relaxation
Raman spectroscopy
Additional Notes Colour pages: 32, 43, 68, 73, 75, 88-89, 91, 93, 115, 118-119, 125, 128

 
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Created: Wed, 16 Nov 2011, 13:52:45 EST by Mr Md. Haque on behalf of Library - Information Access Service