Effect of UHT processing and microconstituent-macromolecule interactions in model liquid systems enriched with insoluble fibre

Kaur, J 2017, Effect of UHT processing and microconstituent-macromolecule interactions in model liquid systems enriched with insoluble fibre, Doctor of Philosophy (PhD), Science, RMIT University.

Document type: Thesis
Collection: Theses

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Title Effect of UHT processing and microconstituent-macromolecule interactions in model liquid systems enriched with insoluble fibre
Author(s) Kaur, J
Year 2017
Abstract Very recently, dietary fibre native to wholegrain cereals attracted considerable interest by consumers and researchers alike due to its demonstrated health attributes, such as lowering the blood cholesterol level and reducing the risk of heart disease. Wholegrain oat contains a variety of microconstituents, mainly dietary fibre, proteins, lipids and phytochemicals (phenolic compounds), which can be beneficial to human health. Because of these advantageous properties, food processors incorporate finely milled oat powder into commercial food formulations along with other commonly added ingredients namely as protein, sugar (sucrose) and vegetable oil. However, understanding how ultra-high temperature (UHT) processing and prolonged storage of up to 8 months at ambient temperatures (20 to 25°C) affect these microconstituents in food products is limited.

Literature contains insufficient data of molecular interactions that can occur between oat microconstituents and various food ingredients following UHT treatment (120-145°C for very short time) as well as prolonged storage. Therefore, this PhD study aims to unveil the effect of UHT processing and prolonged storage on the bioactivity of oat microconstituents, and to investigate the nature of interactions occurring between these microconstituents and commonly added ingredients in model UHT beverages. This will be achieved by employing numerous analytical and physicochemical techniques, i.e. liquid- and gas-chromatography, Fourier transform infrared (FTIR), fluorescence, UV-vis and circular dichroism spectroscopy, particle size analysis, surface charge analysis, dynamic interfacial tension, molecular docking, and quantum energy calculations.

In the first experimental chapter, the influence of UHT treatment (145°C for 8 s) and storage temperatures (22 and 40°C) on phenolic compounds (phenolic acids and avenanthramides), free fatty acids and volatile secondary lipid oxidation products in model oat-beverage formulations containing various concentration of oat powder were examined over 12 weeks. Ferulic acid, followed by p-coumaric acid, was found to be the most abundant phenolic acid in all beverage formulations containing oat. Major avenanthramides (namely A, B and C varities), free fatty acids (palmitic, linoleic and oleic), and volatiles (hexanal and 2-pentyl furan) were also detected from chromatographic analysis.

It was reported that following UHT treatment, the total phenolic content (TPC), phenolic acids (particularly p-coumaric and ferulic acids), free fatty acids and avenanthramides decreased. However, prolonged storage of the UHT-treated preparations at ambient temperature had a positive effect on the content of TPC, phenolic acids, avenanthramides, free fatty acids and volatile compounds. In contrast, extended storage at the higher temperature of this study (40°C) decreased TPC and phenolic acid content; the levels of avenanthramides and volatiles increased with time. Overall, UHT treatment and storage conditions had a substantial effect on the composition of bioactive compounds in oat samples of industrial interest.

The second experimental chapter examined the molecular interactions and bioactivity of added food ingredients in model oat-based beverages, following UHT processing, at 22°C and for a storage period of twelve weeks. Oat particle concentration of 5% (w/w), sucrose (6.7% w/w), vegetable oil (1.8% w/w) and skim milk powder (2.8% w/w), were utilised to create a number of model beverage formulations with commercial relevance. Results indicate that the insoluble dietary fibre of oat particles contains the majority of phenolic acids, with ferulic acid comprising the largest proportion followed by p-coumaric acid. Application of UHT processing decreased the content of individual phenolic acids (ferulic and p-coumaric acids).

In contrast, the level of aforementioned chemical moieties increased with extended storage at 22°C following UHT processing. Among model beverages, those with added milk protein demonstrated a considerable loss of phenolic acids due to the interaction between these micronutrients and milk protein. The nature of molecular interactions was mainly categorised as covalent, with hydrogen bonds playing a supportive role. UHT processing of oat-based beverage formulations facilitated the formation of protein-phenolic acid complexes, which were largely covalent and thermodynamically static in nature. These findings underlined the ability of UHT processing to induce chemical intercations of food ingredients in complex liquid systems.

The third experimental chapter examined the significance of UHT processing and storage conditions, at two temperatures (22 and 30°C) for twelve weeks, on the evolution of free fatty acids and lipid oxidation products from oat grains. Work was extended to molecular interactions with other added ingredients. In doing so, model liquid foods of industrial interest were designed utilising finely milled oat particles, skim milk powder, sucrose and vegetable oil. Three major free fatty acids, i.e. palmitic, oleic and linoleic acids were detected across the entire range of preparation.

Processing and storage conditions led to the development of major lipid oxidation products i.e. 2-pentyl furan and hexanal. Storage temperature variation from 22 to 30°C had a minor effect on the composition of these microconstituents. However, twelve-week storage exhibited an increase in the level of free fatty acids and secondary oxidation volatiles at both storage temperatures. Addition of milk protein reduced the detected content of free fatty acids and volatiles due to direct interactions among these materials. The molecular nature of the interaction between added milk protein and lipid components of oat grain is of importance for the organoleptic consistency of beverage product concepts. These were examined in some detail to reveal a strong covalent nature in the association between milk protein and free fatty acids.

Given the preponderance of covalent interactions between added milk protein in formulations and microconstituents of oat grain, the fourth experimental chapter of this investigation was designed to obtain further insights into their molecular nature. Monodisperse materials were used, i.e. β-casein, which is a major milk protein, and p-coumaric acid at the experimental temperature of 145°C, to identify the specific chemical moiety of the protein responsible for this type of interaction. Analysis focused on spectroscopic fluorescence quenching that provided the Stern-Volmer quenching constant (Ksv), the number of binding sites (n), binding constant (Kb), and the thermodynamic parameters of the free energy of heterotypic association.

p-Coumaric acid complexation altered the secondary structure of the protein at the elevated temperature of our treatment by reducing the extent of α-helix and β-sheet conformation thus opening the protein structure to accommodate the ligand in close proximity. Molecular modelling revealed that the probable binding sites of p-coumaric acid are located in the hydrophobic domain of β-casein, where the p-coumaric acid moiety is further linked to the isoleucine27 residue via a hydrogen bonds (2.85 Å). Quantum energy calculations were performed to predict the covalently reacting amino-acid residue with the ligand, and it was concluded that lysine47 can potentially establish a covalent bond (1.50 Å) with p-coumaric acid following UHT processing. This is an entirely unexpected result, since the literature has been arguing mainly for non-covalent interactions between milk proteins and phenolic compounds at neutral pH and ambient temperature.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Science
Subjects Food Chemistry and Molecular Gastronomy (excl. Wine)
Keyword(s) Insoluble fibres
UHT processing
Protein-phenolic acid covalent interactions
Effect of temperature
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Created: Wed, 21 Feb 2018, 08:05:06 EST by Denise Paciocco
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