Fundamental studies of the effect of glass transition temperature on enzymatic activity in high-solid biomaterials

Chaudhary, V 2013, Fundamental studies of the effect of glass transition temperature on enzymatic activity in high-solid biomaterials, Doctor of Philosophy (PhD), Applied Sciences, RMIT University.


Document type: Thesis
Collection: Theses

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Title Fundamental studies of the effect of glass transition temperature on enzymatic activity in high-solid biomaterials
Author(s) Chaudhary, V
Year 2013
Abstract This PhD research utilizes our understanding of the molecular dynamics of carbohydrates at the vicinity of the glass transition temperature (Tg) with the aim to enhance the stability and quality of processed foods where enzymatic processes are critical considerations. In this context, enzymatic activity in relation to storage temperature addresses the issue of diffusion-controlled substrate/enzyme interactions. Attempts have been made in the literature to follow the rates of enzymatic reactant consumption but were unable to follow convincingly the kinetics of molecular processes within the glass transition region. Our work indicates that the so-called “universal” C1 and C2 values of the free volume theory should not be employed in fundamental research that probes molecular property, since they cannot describe adequately the structural complexity or the specific free volume of a given bio-macromolecule. Problems encountered with this type of approach include negative estimates or physically unrealistic high values of free volume.

To advance fundamental understanding in this field, we developed carbohydrate or protein matrices based on distinct patterns of glycosidic or peptide linkages found in deacylated gellan, gelatinised starch and whey protein isolate (WPI). Characterisation techniques included small-deformation mechanical spectroscopy, micro and modulated differential scanning calorimetry, light and scanning electron microscopy, Fourier transform infrared spectroscopy and wide angle x-ray diffraction. Experimental results of the vitrification properties in the various polymeric matrices, obtained using these physicochemical techniques, were modeled on the basis of a combined theoretical framework of the time-temperature superposition principle, mechanical shift factor, reaction rate theory and free volume theory. This framework of analysis was able to produce fundamental values of the glass transition temperature, as the threshold of two distinct molecular processes, and pinpoint specific C1 and C2 values of the free volume theory for polymeric matrix.

Once the physics of the polymeric material within the glass transition region were understood, we used directly the glassy matrix as the substrate in gelatinised starch/α-amylase preparations or designed non-metabolisable glassy matrices to incorporate the reactive system of pNPG/α-glucosidase in deacylated gellan and pNPG/α-glucosidase in whey protein isolate. A UV-visible spectrophotometric procedure was adapted to analyse the hydrolytic activity of α-glucosidase on pNPG within the condensed gellan or WPI systems, whereas the activity of α-amylase on starch was monitored using reducing sugar analysis with a dinitrosalicylic acid assay. In a further addition to this field, the work introduced to the literature the concept of spectroscopic shift factor as a means of estimating the energy of activation associated with enzymatic hydrolysis of the substrate in vitrified systems. Results strongly argue for a pronounced effect of the gelling bio-macromolecule on enzymatic activity near the mechanical Tg of the matrix, and that the mechanical Tg should be considered and utilised as an effective tool in the quality control and development of novel formulations with desirable structural property and bio-functionality.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Applied Sciences
Keyword(s) Glass transition temperature
enzymes
starch
gellan
whey protein
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Created: Tue, 04 Jun 2013, 10:29:25 EST by Brett Fenton
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