School of Chemistry - Theses
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ItemUltrasound-induced Inactivation of Trypsin Inhibitors for Improving their Functionality( 2023-08)Trypsin inhibitors are anti-nutritional proteins that hinder the digestibility of legume proteins in the gastrointestinal tract, therefore, limiting the application of raw legumes and the consumption of legume products. To improve the commercial and nutritional values of legume products, it is vital to inactivate the two trypsin inhibitors, Kunitz inhibitor and Bowman-Birk inhibitor. However, the traditional thermal inactivation process has unsatisfactory inactivation performance due to the high heat and pH stability of trypsin inhibitors. Therefore, some advanced technologies with high-efficiency and energy-saving should be considered to achieve more effective inactivation of soy trypsin inhibitors. In this thesis, both low- and high-frequency ultrasound treatments were applied to inactivate the Kunitz and Bowman-Birk inhibitors, both in the aqueous phase and in emulsions consisting of the aqueous and non-aqueous phases. The mechanism of ultrasound-induced inactivation and ultrasound-assisted interfacial adsorption and inactivation of soy trypsin inhibitors were proposed and the effect of process-relevant parameters on the ultrasound-assisted inactivation was explored. Additionally, the numerical simulation was used to clarify the mass transfer behaviour of ultrasound-assisted soy amino acid adsorption.
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ItemSonosynthesis of Functional Micro/Nano-structures using Biomolecules( 2022)Malnutrition and access to affordable health services are some of the world’s most urgent problems. The development of nutrient and drug delivery systems by using sub-micro particles as carriers has attained much attention for improving the nutritional value of food and the efficacy of diagnostic/therapeutic treatments. Common methods for synthesizing biofunctional particles usually require many reagents and involve multiple steps. In this regard, a novel and advanced approach for material synthesis needs to be developed and investigated to address those limitations. Ultrasonic techniques have emerged as one-pot and eco-friendly methods for the synthesis of organic and inorganic materials. It has been found that acoustic cavitation derived from sound waves can induce emulsification of liquids to form microcapsules and promote chemical modifications of biomolecules. Protein-shelled microcapsules have been synthesized by using low-frequency ultrasound and reducing agents for food-based applications. However, the direct use of food ingredients for the microencapsulation of microcapsules without resorting to additional external agents was never explored and needs to be investigated further for food-based applications. On another note, drug carriers are usually prepared in nanoscale to enhance interaction with cell membranes for achieving efficient therapeutic treatment. Conventional strategies for preparing drug loaded nanoparticles require matrix materials as carriers, resulting in low drug loading capacity and safety issues. Therefore, synthesis of nanodrugs solely made of antibiotic molecules is a better method for development of drug delivery platforms. Many molecules bearing aromatic groups have been successfully sono-assembled into nanoparticles by high-frequency ultrasound, but they are mainly used as drug carriers. Transforming drug molecules into carrier-free nanodrug has not been widely investigated. As such, I intend to expand new research towards other drug molecules with aromatic moieties. In this regard, my Ph.D. project aims to sono-chemically synthesize various micro and nano structures from biomolecules by tuning the frequency/power of ultrasound without the usage of external reagents. The size of the obtained bio-functional structures is controllable, and their compositions are suitable for use in specific applications such as : i) nutrients delivery in food industries; ii) drug delivery for biomedical applications. The fundamental concepts of sono-chemistry for material synthesis, along with biomolecules (proteins, nutrients and antibiotics) based micro/nano structures and their applications are discussed in Chapter 1. Chapter 2 provides an overview of microencapsulation techniques for food industries and fabrication of nanoparticles for antibiotics delivery. In particular, methodology, formulating materials, current challenges, limitations and innovation are discussed. In Chapter 3, the materials, equipment and methodologies involved in the reactions used in this thesis are thoroughly described. Chapter 4 is the first chapter of result and discussion section. Microcapsules made of egg white protein (EWP), as commonly available biopolymers, were first conceptualized. Oil-soluble nutrients (Vitamin A, D and E) were encapsulated into EWP to form nutrients loaded proteinaceous microcapsules by employing 20 kHz ultrasound. This work primarily points out that high availability of free thiol groups in protein solution is crucial in forming stable microcapsules with robust shells, in order to protect micronutrients from degradation against detrimental effects. In Chapter 5, another two plant-based protein isolates extracted from soybean (SPI) and corn (CPI) were also formulated to form microcapsules. This study provided further insights into the structural, chemical and surface properties of proteins for efficient ultrasonic microencapsulation of micronutrients. A double emulsion technique was further developed to co-encapsulate both oil- (vitamin A and D) and water-soluble (vitamin B, C and minerals) micronutrients. In-vitro digestion study showed that the proteinaceous microcapsules enable sustained release of micronutrients, demonstrating their potential in food fortification applications. In Chapter 6, a sono-chemical strategy for transforming antibiotic doxycycline into carrier-free nanodrugs via high-frequency ultrasound (490 kHz) is reported. This study demonstrates that doxycycline undergoes hydroxylation and dimerization processes upon sonication in an aqueous solution to ultimately self-assemble into nanoparticles. The size of obtained particles could be finely controlled by tuning the applied ultrasonic powers. The nanodrugs exhibited antioxidant properties, along with antimicrobial activity against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacterial strains. These results highlight the feasibility of the ultrasound-based approach for engineering carrier-free nanodrug with multiple controlled bio-functionalities. Chapter 7 provides an overall summary of the entirety of my PhD project as well as my conclusion and thoughts on it.
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ItemThe effects of ultrasound on the molecular, structural and nutritional aspects of dairy proteins( 2021)Process improvement and product improvement in the dairy processing industry is an ongoing dynamic process. Ultrasound processing technology for food processing is in its developmental stages, and dairy sector is a key area for potential applications. The use of low frequency and high frequency ultrasound to modify functional properties of dairy streams is being widely studied by seeking to modify the protein and lipid components of milk. The effects of ultrasound on size of lipid globules and lipid oxidation volatiles have been studied. The effects on functional properties of milk proteins such as viscosity, gelation, heat stability etc. have been documented. However, the present literature lacks information on the effects that ultrasound processing may have on the fundamental properties of dairy proteins. Therefore, it is of importance to document the effects of ultrasound processing on the molecular, structural and nutritional aspects of dairy proteins. As such, the effects of ultrasound processing on dairy proteins have been studied in the context of the amino acid composition, amyloid modifications, and bioactive peptides released. This work aims to bridge the gap in existing literature attempting to illuminate upon these fundamental aspects. Chapter 1 in this thesis introduces the premise of this research by discussing the fundamental properties ultrasound technology and presents dairy proteins as the central theme of this research. A systematic review of literature in Chapter 2 attempts to build familiarity with the effects of processing on milk proteins; and discusses the potential applications of ultrasound in the dairy industry – noting the studies done on whey are caseins. This chapter also discusses the research areas which could be addressed to answer questions related to the objectives of current PhD work. To encourage the application of ultrasound for dairy processing, it would be of significance to understand the extent of operating parameters that ensure nutritional integrity and food safety. The amino acid integrity of skim milk proteins after sonication along with the potential of ultrasound to modify secondary structural conformations of milk proteins has been demonstrated in Chapter 4. Chapter 5 expands the objectives of Chapter 4 into whole milk systems, taking into account milk lipids and the potential for oxidation of lipids on sonication. In Chapter 5 an effort has been made to better understand the underlying cause of lipid oxidation observed due to ultrasound processing of milk lipids. The observations from this study suggest that the enhanced mass transfer of oxygen due to ultrasonic shear forces makes oxygen non-limiting in lipid phase and increases auto-oxidation reactions. An assessment of amino acid integrity of full cream milk after ultrasound treatment confirmed oxidative stability of milk proteins. These findings complement the data in Chapter 4. Along with amino acid integrity, Chapter 4 also demonstrates that the secondary structural conformations of milk proteins can be modified by ultrasound. The work in Chapter 6 builds up greater understanding on such changes by studying the effects of low frequency ultrasound on isolated beta-lactoglobulin (b-lg). b-lg is a b-sheet rich protein with amyloidogenic potential. The effects of high shear and high temperature microenvironments produced by ultrasonic waves were studied on b-lg aggregation. Ultrasound induced formation of amyloid crystals in dilute b-lg solutions at neutral pH and ambient bulk temperatures. The theoretical reasoning for the aggregation phenomenon substantially adds to the understanding of protein energy landscape. Chapter 7 focusses on nutritional properties of milk proteins. It examines the effect of ultrasound pre-treatment on the release of BCM-7 bioactive peptide from milk by simulated gastro-intestinal digestion (SGID), and the effect on overall protein digestibility. It demonstrates that ultrasound processing could be used to downregulate the release of undesirable BCM-7 bioactive peptide in milk, by affecting the rate of enzymatic hydrolysis of certain intermediate peptide fragments. The findings from this body of work add to the existing knowledge for improved use of ultrasound to ensure nutritional integrity of dairy proteins and lipids, minimising oxidative damage. It also builds on current understanding of ultrasound induced protein restructuring and its potential for modification of peptide release from dairy proteins.
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ItemUltrasonic emulsification in biopolymer complex emulsion systems( 2019)An emulsion is a colloidal mixture of two immiscible liquids, in which one is dispersed into another with the help of shear forces and the presence of surface-active compounds. The range of applications for emulsions is enormous, with emulsions found in industries such as food, cosmetics, medicine and petrochemicals. Understanding how to control the physical properties of emulsions has been a long-standing research challenge. In particular, the ability to achieve long-term stability of emulsions is of great significance in many practical applications. The physical stability of emulsions can be improved with a reduction in the droplet size of the dispersed phase, which is determined by the extent and intensity of the emulsification process and the associated balance between droplet breakup and coalescence. High-intensity low-frequency ultrasound (US) is a promising high-shear emulsification method that leverages the physical effects created by acoustic cavitation. Biopolymers are naturally occurring polymers that have many applications in food and personal care products. In particular, biopolymer emulsions are found in many daily products. The ability to tailor the physical properties of such emulsions, including texture, stability and flow behaviour, is a focus of much research and development. Emulsions are commonly fluid suspension, however they can also undergo a solution-gel transition, greatly altering the macroscopic texture into a semi-solid material. Emulsion gel formation has been reported in the literature, however, little is understood about the role of the emulsification process, in particular, associated turbulent flow, on the mechanisms on emulsion gel transition. In this thesis, ultrasonic emulsification involving complex biopolymer systems has been studied. The thesis presents new fundamental understandings of this process, in particular the effect of the turbulent environment on biopolymer emulsion gel formation. This understanding has been used to develop a novel type of emulsion gel, for which the formation mechanism has been investigated and described. A novel demulsification technology has been also developed for enhanced oil separation from biomass systems. The basic concepts underlying emulsions, emulsification and biopolymers are introduced first introduced in Chapter 1 along with fundamentals of US and sonochemistry. In Chapter 2, a detailed review of the literature is presented describing the current understanding of emulsification in turbulent flows, the mechanism and control of ultrasonic emulsification, the mechanism of colloidal sol-gel transition in biopolymer emulsion systems, and flow-induced phase inversion. Additional attention is given to literature on casein micelles and dairy emulsion, as this was the system explored in most detail experimentally in the thesis. Knowledge gaps in the literature are identified and the research aims and thesis scopes placed into this context. The experimental details, including materials, methodologies and analytical techniques are presented in Chapter 3. The experimental research results are presented and discussed in Chapter 4 to 6. In Chapter 4, three essential experimental parameters in the ultrasonic emulsification process, namely sonication time, acoustic amplitude and processing volume, are individually investigated, theoretically and experimentally, and correlated to the emulsion droplet sizes produced. The results show that with a decrease in droplet size, two kinetic regions can be separately correlated prior to reaching a steady-state droplet size: a fast size reduction region and a steady-state transition region. In the fast size reduction region, the power input and sonication time can be correlated to the volume-mean diameter by a power-law relationship, with separate power-law indices of -1.4 and -1.1, respectively. A proportional relationship is found between droplet size and processing volume. The effectiveness and energy efficiency of droplet size reduction has been compared between US and high-pressure homogenisation (HPH) based on both the effective power delivered to the emulsion and the total electric power consumed. Sonication could produce emulsions across a broad range of sizes, while high-pressure homogenisation is able to produce emulsions at the smaller end of the range. For ultrasonication, the energy efficiency is higher at increased power inputs due to more effective droplet breakage at high US intensities. For HPH the consumed energy efficiency is improved by operating at higher pressures for fewer passes. At the laboratory scale, the US system requires less electrical power than HPH to produce an emulsion of comparable droplet size. The energy efficiency of HPH is greatly improved at large scale, which may also be true for larger-scale ultrasonic reactors. In Chapter 5, shear-induced emulsion gel formation has been demonstrated for the first time in a micellar casein emulsion system subjected to micro-turbulent environments created by ultrasonication and high-pressure homogenisation. Importantly, the emulsion and gel-like properties are stabilised solely by the casein micelles in combination with the droplet packing structure, circumventing the usual requirement for chemical surfactants and stabilisers. The mechanism of shear-induced emulsion gel formation has been investigated experimentally in relation to the roles of casein micelles, oil fraction and shear environments, and discussed in relation to existing theories. Based on this, the mechanism of gel formation has been proposed as a novel colloidal packing phenomenon, triggered by the formation of droplet breakup under micro-turbulent environments, which results in fractal packing of droplets and casein micelles over three orders of magnitude from 10-1 to 101 micron. In Chapter 6, the proposed mechanism underlying the shear-induced formation of casein micelle emulsion gels has been extended to other systems by providing a more general explanation on a fundamental level. The effect of micellar casein concentration on the sol-gel transition is investigated to reveal a shear- and concentration-dependency and non-monotonic rheological behaviour. A successful imaging protocol is developed to examine the in-situ interfacial rearrangement of micellar casein. The method involved substituting food oils with a volatile solvent, so that both the oil phase and water can be removed and the micellar bridging network inspected in detail by scanning electron microscopy. A large degree of interfacial deformation of micellar casein has been observed, from spheres to discs, which were further inter-connected to form cellular protein 3D networks. The combination of emulsion microstructure and the rheological profiles could be simply explained by the established sol-gel transition models in colloidal suspension systems. Rather, these experimental results are explained using a combination of two theories: random Apollonian packing and droplet size distribution scaling under turbulent flows. The existence of a sol-gel transition could be related to power-law correlations in the number size distribution of the droplets within a certain size range. The results confirm that the casein micelles and emulsion droplets could be unified as ‘spheres’ participating collectively in random close packing. The mechanism of sol-gel transition under turbulent flows has been proposed more generally as a result of turbulence-driven dynamic changes in the number size distribution of ‘spheres’. The formation of shear-induced emulsion gels has been then further extended with key criteria proposed, based on the new multi-theory mechanism framework. The understandings gained in this thesis are summarised and concluded in Chapter 7. Future research directions are also discussed based on these new findings. Highlights of two commercialisable extensions of the work of the thesis are also provided as supplementary material in Appendices A and B.
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ItemUltrasound driven synthesis of bio-functional nanostructures( 2019)Polyphenolic-, amino acids- and doxorubicin-based nanostructures are of great interest due to their multifarious applications in biomedical field as antibiotics, antioxidants, antimicrobial and anti-cancer agents. Most of the research on the polyphenolic and amino acids based nanostructures adhere to the formation of coordination complexes with metals, self-assembly techniques or chemical functionalization and crosslinking reactions. To improve the efficacy of therapeutic agents, a variety of nanoparticles have been developed for the controlled and targeted delivery of doxorubicin. These include biopolymers-based nano/microcapsules, carbon-based nanoparticles, polymer-drug conjugate, dendrimers, liposomes, micelles, inorganic nanoparticles and nucleic acids nanostructures. The development of simple, one pot and effective synthesis routes to fabricate bio-nanomaterials is in high demand. In particular, the use of polyphenols, doxorubicin and single amino acids as building blocks to fabricate nanostructured materials is still unexplored. In this PhD work, I have used ultrasound-based technologies to synthesize phenolic, amino acid and doxorubicin based micro and nanoparticles for different biomedical applications. Chapter 1 provides an overview on the structural and bio-functional properties of nanoparticles and methods to synthesize nanoparticles for biomedical use, including the ultrasonic techniques have been discussed. Furthermore, fundamentals of ultrasound are also provided. In the literature review (Chapter 2), several studies dealing with the polyphenol, doxorubicin and amino acid molecules have been summarised. In the last section of this chapter, numerous investigations on synthesis of diverse types of nanostructures using ultrasound are reviewed. In Chapter 3 materials and methods, equipment and all other experimental details used are described. Chapter 4 provides a fundamental understanding on the ultrasonic coupling of simple phenolic molecules, where acoustic bubble acts as a catalytic binding site for the generation of bioactive oligomers without the need for utilizing any enzymes, catalysts (organic or inorganic) and other toxic reagents. It has been observed that the extent of oligomerization and nanoparticles formation depends on the ultrasonic frequency, concentration and physiochemical properties of the phenolic building blocks. Chapter 5 demonstrates that cavitation bubbles are simple micro-reactors with reactive surfaces to perform one-pot multiple reactions on complex polyphenolic molecules to convert tannic acid to ellagic acid, namely (i) hydrolysis of an ester linkage, (ii) C–C coupling reactions, (iii) condensation reactions and (iv) crystallization of the product into regularly shaped particles. The size and shape of the crystals can be controlled by ultrasonic frequency, power and time. The synthesized particles exhibit fluorescence properties, anticancer and antioxidant activity and could be further used for drug loading and delivery. In Chapters 6 and 7, the role of the acoustic field in the formation of supramolecular nanoaggregates using tryptophan and phenylalanine as building blocks was investigated. It has been demonstrated that the acoustic bubbles driven at high frequency standing wave, in addition to provide a reactive surface for the dimerization of biomolecules, can also provide an energy source to fuel and refuel the dissipative out of equilibrium assembly of these molecules below the critical aggregation concentration. Furthermore, the unique optical and bio-functional properties of nanoparticles for bioimaging and probing the intracellular trafficking of a drug have also been studied. In Chapter 8 the sound-driven self-assembly of the anticancer drug doxorubicin was investigated to generate nanoparticles solely made of drug molecules. The newly developed nanoparticles were tested on different types of cancer cells and the drug was found to be active in drug resistant cell lines. In addition, the mechanism of action of drug nanoparticles was investigated. Chapter 9 provides a summary of the entire PhD work.