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    Effect of air temperature on the thermal degradation of heat liable products in spray drying and monodisperse drying : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Manawatū, New Zealand
    (Massey University, 2018) Sang, Xiaoqi
    Three heat liable protein-based materials β-galactosidase, whey protein isolate (WPI) and egg white (with 30-35% w/w total solids) were dried through conventional spray drying and monodisperse drying respectively with constant inlet air temperature 200 ℃ and different outlet air temperature. The purpose was to test the hypothesis that monodisperse droplet drying could produce more control over time-temperature experience during drying, resulting in reduced loss of structure or activity. The residual enzyme activity of the dried lactase product was determined by ONPG β-galactosidase assay, and the extent of denaturation of WPI and egg white was determined by differential scanning calorimetry (DSC). Particle size and morphology were also measured and observed. The results showed that for both spray drying and monodisperse drying, the extent of protein denaturation increased as outlet air temperature increased. In comparison with spray drying, monodisperse drying had a longer residence time using our particular apparatus and gave rise to higher extent of heat degradation for all three materials. The dried products from monodisperse drying showed a narrower particle size distribution but had larger particle size compared to the products from spray drying. The majority of monodisperse dried powders had a multivesicular hallow morphology due to high interior temperature and coalescence of neighbouring particle in flight. The feasibility of using monodisperse drying in real industry is still under investigation. Although the results obtained from this study denied the expectation that monodisperse drying can reduce the thermal degradation of product during drying process, they are still useful in developing the monodisperse drying system and optimizing the operating parameters.
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    β-Lactoglobulin nanofibrils: Effect of temperature on fibril formation kinetics, fibril morphology and the rheological properties of fibril dispersions
    (Elsevier Ltd, 2012-05) Loveday SM; Wang XL; Rao MA; Anema SG; Singh H
    Almost all published studies of heat-induced b-lactoglobulin self-assembly into amyloid-like fibrils at low pH and low ionic strength have involved heating at 80 C, and the effect of heating temperature on self-assembly has received little attention. Here we heated b-lactoglobulin at pH 2 and 75 C, 80 C, 90 C, 100 C, 110 C or 120 C and investigated the kinetics of self-assembly (using Thioflavin T fluorescence), the morphology of fibrils, and the rheological properties of fibril dispersions. Self-assembly occurred at all temperatures tested. Thioflavin T fluorescence increased sigmoidally at all temperatures, however it decreased sharply with >3.3 h heating at 110 C and with >5 h heating at 120 C. The sharp decreases were attributed partly to local gelation, but destruction of fibrils may have occurred at 120 C. Thioflavin T fluorescence results indicated that maximal rates of fibril formation increased with increasing temperature, especially above 100 C, but fibril yield (maximum Thioflavin T fluorescence) was not affected by temperature. At 100 C and 110 C, fibrils were slightly shorter than at 80 C, but otherwise they looked very similar. Fibrils made by heating at 120 C for 1 h were also similar, but heating at 120 C for 8 h gave predominantly short fibrils, apparently the products of larger fibrils fragmenting. Heating at 100 C gave consistently higher viscosity than at 80 C, and heating for >2 h at 120 C decreased viscosity, which may have been linked with fibril fragmentation seen in micrographs.
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    Effects of environmental factors on heat-induced [beta]-lactoglobulin fibril formation : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Food Technology, Riddet Institute, Massey University, Palmerston North, New Zealand
    (Massey University, 2010) Wang, Xiangli
    The heat-induced fibrillar aggregation of β-lactoglobulin was studied under various environmental conditions. The formation of β-lactoglobulin fibrils was monitored by Thioflavin T (ThT) fluorescence and their morphology was studied using transmission electron microscopy (TEM). Amyloid-like fibrils were formed under standard conditions (pH 2.0, 80°C and low ionic strength). The β-lactoglobulin fibrillation kinetics exhibited sigmoidal behaviour, and the two-step autocatalytic reaction model fitted ThT fluorescence data well. The studies of the individual effect of pH, temperature, NaCl, CaCl2 on β-lactoglobulin fibril formation showed that decreasing pH (2.4 - 1.6), increasing temperature (75 - 120°C) and increasing salt concentration (NaCl 0-100 mM; CaCl2 0-100 mM) accelerated the fibril formation process and altered the morphology of fibrils. The two-step autocatalytic reaction model did not fit the ThT fluorescence data well at higher temperature (>100°C) or at low pH (1.6). The effects of the four factors (pH, temperature, NaCl and CaCl2) on β-lactoglobulin fibril formation were studied by using a central composition design (CCD) experiment. Results showed that the four main and some of the non-linear effects were significant (95%) on fibril formation, including fibrillation time and the fibril yield. Taking all results together, it can be implied that β-lactoglobulin fibril formation can be promoted by choosing the external incubation conditions. This study is the first step towards the application of protein fibrils as texture-modifying ingredients in food systems.
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    Effect of bound ligands and [kappa]-casein on the denaturation of [beta]-lactoglobulin : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University
    (Massey University, 2000) Cho, Younghee
    The objective of this study was to gain greater understanding of the behaviour of bovine β-lactoglobulin (β-lg) during thermal denaturation. The first part of the study was focused on gaining more knowledge of the structural changes in β-lg during heat-induced and urea-induced denaturation and the effect of ligands and the genetic variants of β-lg. The second part of the study explored the mechanism of the heat-induced interaction of β-lg with κ-casein (κ-CN) and the effects of genetic variants. The reversible early steps during thermal denaturation of β-lg are not readily separated from the later irreversible steps which involve sulphydryl-disulphide interchange reactions. However, it should be possible to examine the behaviour of β-lg in the early steps without reactive thiols either by using a porcine β-lg, that does not have cysteine residue equivalent to Cys-121 of bovine β-lg, by blocking the free thiol or by using solvent denaturation. Heating to 80 °C irreversibly altered near- and far-UV circular dichroism (CD) spectra, and 1,8-anilinonaphthalene sulphonate and retinol binding fluorescence spectra for bovine β-lg, but not for porcine β-lg which unfolded reversibly. Also the examination of the induced CD spectral changes of retinol and cis-parinaric acid (PnA) on binding to bovine β-lg B upon heating revealed that bovine β-lg lost the ability to bind retinol and PnA in a chiral environment. In contrast, porcine β-lg did not show significantly induced CD bands upon mixing with retinol, but PnA/β-lg mixtures showed induced CD bands of low intensity. In addition to the lack of a free thiol group in porcine β-lg, the sequence differences between bovine β-lg and porcine β-lg are also likely to affect the behaviour of these β-lgs during heat treatment and the binding of ligands. Although palmitic acid does not show any absorbance in CD spectra, it enhanced the stability of the bovine β-lg and porcine β-lg mixture. The urea-induced unfolding of bovine β-lg at neutral pH (6.7) revealed that the stability of the genetic variants followed the order, β-lg B<β-lg A<β-lg C, as observed during thermal denaturation and tryptic hydrolysis. The stabilisation effect was also observed by adding retinol, retinoic acid and palmitic acid during urea denaturation of β-lg, and by retinol, retinyl acetate and PnA during ammonium sulphate denatuation of β-lg. Blocking the sulphydryl group of β-lg destabilised the native protein against urea denaturation through the introduction of a bulky group to the compact structure of β-lg. This result, together with the results for porcine β-lg, confirms that the sulphydryl group plays an important role in the unfolding of bovine β-lg. In the second part of the study, an attempt was made to investigate the effect of bovine κ-CN on the established heat-induced unfolding and aggregation pathway of bovine β-lg, by adding κ-CN A or κ-CN B to native or pre-heated β-lg A, B or C and heating the mixture. The CD band intensity of the mixture of β-lg and κ-CN at 270 nm, an index of significant alteration to the disulphide bond dihedral angle, indicated increasing structural changes involving disulphide bonds during heat treatment. The rates of loss of β-lg and the distributions of intermediate products were determined using alkaline- and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). During reaction with β-lg, some monomeric κ-CN was found by SDS-PAGE, probably as a result of disulphide bond interchanges between κ-CN and β-lg, and two-dimensional PAGE also revealed disulphide-bonded β-lg/κ-CN aggregates. In the presence of κ-CN, the loss of monomeric β-lg increased and less non-native monomer and dimer were observed compared to β-lg alone and κ-CN reacted more rapidly with β-lg that had been unfolded by prior heat treatment than with native β-lg. This suggested that β-lg probably denatured (unfolded) independently and either simultaneously or consequently and contained higher sulphydryl reactivity than native β-lg, which lead to the reactions with κ-CN via disulphide bond interchange. It is possible that the equilibrium between native β-lg and denatured β-lg shifted rapidly, because κ-CN preferred to interact with denatured (unfolded) β-lg that has higher sulphydryl reactivity than native β-lg. The kinetics of the interaction between β-lg and κ-CN were evaluated from the heat-induced loss of alkaline-monomeric β-lg at 80 °C. The interaction between β-lg and κ-CN could not be described by any reaction order between 1.0 and 2.0. The slopes of the plots changed at about 7.5-10 min heating time and this corresponded to intensity changes in the alkaline-monomeric, non-native monomeric and dimeric β-lg bands, which increased during the first 10 min of heating and then slightly decreased or remained relatively constant for the rest of the heating. The loss of native β-lg in β-lg/κ-CN mixtures during heating at 80 °C was shown to be significantly influenced by the genotypes of both β-lg and κ-CN. The κ-CN B variant showed considerably higher reactivity than κ-CN A, while the β-lg B variant was the most reactive. The greatest loss of native β-lg was observed from the β-lg B/κ-CN B mixture