CORA
Cork Open Research Archive (CORA) is UCC’s Open Access institutional repository which enables UCC researchers to make their research outputs freely available and accessible.
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Impact of protein genotypes on milk composition and processability
(University College Cork, 2024) Gai, Nan; Kelly, Alan; O’Regan, Jonathan; Goulding, David A.; Uniacke-Lowe, Therese; Nestlé
Milk protein genotypes are associated with differences in milk yield, composition, and processability due to direct effect of their structural differences, or their indirect effects on casein micelles, as well as differences in milk composition. This study investigated the effects of β-casein (β-CN) genotypes on milk physicochemical properties, functionalities, processability (Cheddar cheese processing), and proteolytic mechanisms. Influences of β-lactoglobulin (β-lg) genotypes on heat-induced whey protein denaturation were also investigated.
The investigation of milk physicochemical properties, including gross composition, mineral content, casein micelle size, zeta potential, polydispersity index (PDI) and fat globule size distribution were initially focused on three main β-CN genotypes, A1A1, A1A2 and A2A2. No significant differences were detected on milk composition between three genotypes, and casein micelle sizes between three genotypes were similar. Better rennet coagulation properties and acid coagulation properties were determined in A1A1 milk, in comparison to A1A2 and A2A2 milk, but differences were not significant. A2A2 milk had smaller fat globule size and better stability than A1A2 and A1A1 milk against creaming.
Cheddar cheese was produced using milk with A1A1, A1A2 or A2A2 β-CN genotypes. A2A2 cheese milk had significantly poorer rennet coagulation properties compared to the other two genotypes, which caused a delay in the cutting step. A1A1 cheese had a lower protein content, while A2A2 cheese had a lower fat content compared to the other two cheeses. Protein contents in both A1A1 and A2A2 cheese whey were higher than that in A1A2 cheese whey. Ripened A1A1 cheese was the softest, and the least fracturable.
Dissociation and interfacial properties of purified A1 and A2 β-CN, obtained from milk with A1A1 and A2A2 β-CN genotypes using microfiltration (MF), were studied. A2 β-CN, on micellization, had smaller particle size than A1 β-CN, and A1 β-CN was more stable over heating and cooling than A2 β-CN. Foam stability and emulsifying ability of A2 β-CN was higher than that of A1 β-CN, while stability of emulsions produced using A1 β-CN was higher than that of A2 β-CN.
The differences between the two β-CN genetic variants A1 and A2 in terms of proteolysis in milk were determined. A2A2 milk had higher plasmin activity than A1A1 milk, and A2 β-CN was more susceptible to plasmin than A1 β-CN. Referring to the different amino acid on sequence position 67, where proline (Pro) is in A2 β-CN and histidine (His) is in A1 β-CN, His67 was determined to be more susceptible than Pro67 in hydrolysis of β-CN.
The influence of β-CN genotypes on milk characteristics was also studied for milk containing minor β-CN genotypes, A1B, A2B, A1I and A2I. Larger casein micelle size, better rennet coagulation and acid coagulation properties were associated with β-CN variant B compared to the I variant. The structural differences between the two genotypes, B and I, has also influenced their proteolysis, and the Arg122 in β-CN B variant was determined to be more sensitive to plasmin-derived proteolysis compared to the Ser122 in the I variant. In addition, the His67-Asn68 bond in the A1 and B variants was more susceptible than Pro67-Asn68 in the A2 and I variants to β-CN proteolysis driven by milk indigenous enzymes, while the activities of Ile66-His67 and Ile66-Pro67 were similar.
The effect of κ-CN genotypes was determined to have a stronger influence on milk casein micelle size and fat globule size than that of β-CN genotypes, where milk with κ-CN A had larger casein micelle and smaller fat globule size than milk with κ-CN B. The acid coagulation and rennet coagulation properties of milk containing the B variant of κ-CN were better than milk containing the A variant, which was possibly due to the effect on casein micelle size and fat globule size.
In the study of whey protein denaturation induced by heating, milk with β-lg A variant was more heat resistant than with the B variant. More denatured whey proteins with B β-lg were likely to interact with casein micelles to form insoluble aggregates compared to the A variant.
In conclusion, the findings in this study suggest that milk protein genotypes are clearly associated with milk characteristics and technological properties, and opened new, related areas such as the potential linkage between specific peptides obtained from proteolysis to milk functional properties. These findings can be used as a reference for further studies, and as guidance for milk selection when dairy products with specific characteristics are required.
This House, a Home? A practice-based analysis of Irish community theatre and its potential for politicisation
(University College Cork, 0023) O'Callaghan, Angela; O'Gorman, Roisin; Woodhouse, Fionn; Gleeson, Eibhlín; Cork Opera House; University College Cork
This practice-as-research project analyses the status of community theatre in a wider Irish context through the creation of headphone theatre piece This House a Home - an audio performance through Cork Opera House which explores themes of inclusion, belonging and homebuilding. The script for This House a Home was co-created over a two-month workshop series with vulnerable adults from Cork City who rarely spent time in Cork Opera House. Using arts-based research techniques in the tradition of verbatim/testimony theatre, these participants were recorded each week as they grew confident to critique the efficacy of Cork cultural spaces in making them feel at home. These recordings, dramaturged together to create an audio tour of Cork Opera House, highlight the ways in which these project participants came to feel at home in the space, and conversely, the ways in which they were further marginalised. Aligning the present with the past through archival research on Cork Opera House’s history, specifically the burning of Cork Opera House in 1955 and its subsequent rebuilding, the piece asks of its audience, through the participants: does this house need to be burned down again, to be rebuilt as a home? This distinction between house and home, discomfort and belonging, destruction and renewal, is further posed in this thesis through a three-pronged analysis of the form of the piece, this workshop series and the writing process. I will therein pose two understandings of community theatre making - a ‘romantic’ perception which prioritises a final product and pre-existing audiences and a ‘radical’ perception which emphasises the process of making (a home) and uplifts the marginalised. In posing these two readings of community theatre and applying them both to the creation of this piece and the wider Irish context of community theatre making (which, I argue, is dominated by a romantic understanding of community theatre), this project demonstrates the importance of fusing the radical with the romantic; breaking down of barriers to inclusion for marginalised people in both theatre making and cultural spaces, starting with Cork Opera House.
Investigation of a novel cell penetrating protein for the delivery of biomolecules
(University College Cork, 2024) Cantillon, Emer A.; Fleming, John V (Eoin); Sanghamitra, Nusrat; CyGenica Ltd.
Cellular delivery of therapeutically valuable macromolecules such as proteins and nucleotides, or genome editing systems such as CRISPR-Cas9, are key to advancing the treatment of many diseases. To make this a therapeutic reality however, the safe delivery of cargo across the cellular membrane remains a key obstacle. Furthermore, the development of molecular tools that target intracellular compartments and tissue specific subtypes is necessary in order to improve the therapeutic efficiency of macromolecules and negate off-target effects. In this context, several viral and non-viral systems have been developed to varying degrees, but there remain problems with immunogenicity, carcinogenesis, toxicity and low in-vivo delivery efficiency (1–3).
Here we describe a protein, termed GEENIE, and identify its novel cell penetration capabilities, demonstrating that it can translocate across the plasma membrane of mammalian cells. We show that GEENIE can penetrate cells in a time dependent manner and can proceed in the presence of serum. Additionally, using a combination of biochemical and pharmacological experiments, we demonstrate that the mechanism of GEENIE uptake is not limited by endocytosis and confirm GEENIE uptake in red blood cells that lack endocytic machinery.
Having demonstrated that GEENIE can cross the plasma membrane, we then extended its application to the delivery of biomolecules. As for other non-viral delivery systems, we first assessed the ability of GEENIE to deliver protein cargo such as fluorescent proteins. Using recombinant DNA technology, we expressed GFP tagged GEENIE chimeras. This strategy allowed for the quick and affordable purification of chimeric proteins, whereafter their cellular uptake was visualized using confocal microscopy. We were able to successfully demonstrate the ability of GEENIE to deliver GFP protein cargo intracellularly.
The delivery of biotherapeutics can often be limited by the off-target effects that cause excessive cytotoxic to healthy, non-diseased cells. The cell specific delivery of therapeutics is therefore an important stipulation for delivery vehicles in order to maximize therapeutic output and reduce off target effects. The development of delivery vehicles that contain a moiety to target and deliver to a specific cell surface receptor is a common strategy to improve cell specific targeting. To this end, we investigated a newly identified peptide that has shown specificity to the HER2 receptor. Incorporating the peptide within GEENIE, allowed for increased cell specificity to HER2 expressing cells (SKBR-3) compared to HER2 negative cells (MDA-MB-468).
Having demonstrated that GEENIE can be targeted to cell specific subtypes, can traverse the cellular membrane and deliver protein cargo, we next evaluated a strategy to deliver biomolecules involved in gene therapies. Gene therapies are an important class of therapeutics that have potential in the treatment of a wide variety of diseases, while also providing an opportunity to progress personalized treatment strategies. Two important groups include nucleic acids, such as siRNA, and gene editing toolkits, such as CRISPR. Employing a genetic engineering approach, we produced two separate GEENIE chimeras, R9-GEENIE and Cas9-GEENIE, to achieve delivery of siRNA or Cas9-sgRNA ribonucleotide protein respectively. Despite our optimization attempts we were unable to produce a functional R9-GEENIE to achieve siRNA delivery and protein knockdown. On the other hand, we demonstrated a potential strategy for the delivery of Cas9-RNPs via GEENIE. Future studies to optimize gene knockout may provide extra insight into whether GEENIE can be used for this strategy, and whether it has clinical potential for the delivery of Cas9.
The use of science in EU environmental decision-making: a measure of legitimacy in environmental regulation
(University College Cork, 2024) Jennings, Rhoda; McIntyre, Owen; Poustie, Mark; Environmental Protection Agency; Irish Research Council
This thesis investigates the role of science and scientific evidence in EU environmental law. It examines the practical interaction and relationship between science and the formulation and application of environmental law. In so doing, it takes a legal regulatory approach to exploring the science-law interface.
The research goes beyond viewing science as a component of evidence-informed policy. It explores whether, and to what extent there is a legal obligation to use scientific evidence in the formulation of EU environmental policy and legislation. This is carried out through an examination of the role attributed to science under the Treaty on the Functioning of the European Union, and by exploring the normative role of science as a source of legitimacy in environmental decision-making.
The thesis adopts a practical approach to investigating the role of science in environmental law. It develops a a typology of the primary EU science advisory bodies that assist the Commission in its work, and feed into pre-legislative debate. It carries out case studies on ambient air pollution and nature conservation legislation, tracing the use of science in the formulation and application of the law, from the travaux préparatoires to the adoption of the final legislation, and the interpretation of the law by the Court of Justice of the European Union.
The research consolidates the exploration of the normative role of science in EU environmental legislation and policymaking, with the actual role of science, by drawing on the concept of legitimacy to develop an analytical framework. This framework is used to discuss more precisely how science is used in the formulation of legislation, and the consequences for the legitimacy of the legislation.
The research indicates that there is a wealth of high-quality science advice in the EU. Shortcomings in the regulatory structure of the science-law interface, however, serve to undermine the functional benefits of science. The thesis proposes preliminary methods for enhancing the use of science in EU environmental decision-making.
Fractionation of β-casein, the formation of complex coacervates between β-casein preparations and lactoferrin, and an assessment of their digestion, technofunctional and biofunctional properties
(University College Cork, 2024) van der Schaaf, Jasper Melle; Kelly, Alan; O'Mahony, James; O'Regan, Jonathan; Goulding, David A.; Fondation Nestlé
Lactoferrin (LF) and β-casein are major human milk proteins that have individually been recognized for their significant roles in infant health and nutrition. However, the impact of their complex coacervation, involving liquid-liquid phase separation, and the behavior of these structures during gastrointestinal digestion remains largely unexplored. This thesis aims to advance the fractionation and use of β-casein as a dairy ingredient and knowledge on the formation of LF:β-casein complex coacervates, their physicochemical properties, and biological functionalities within the context of infant in vitro gastrointestinal development.
The commercial production of β-casein is limited, due to challenges with yield, associated costs, and industrial application of the final ingredients. Combining fractionation treatments may improve the overall composition and purity of these ingredients. A novel approach for β-casein isolation was developed in Chapter 1, that included membrane filtration combined with chymosin treatment, which removed the κ-casein fraction from the final enriched β-casein ingredient.
The second chapter focused on the factors influencing the formation of complex coacervates, including pH, ionic strength, stoichiometry, temperature, protein concentration and protein profile, and demonstrated a strong interdependence of these factors. The coacervate yield varied based on the starting material, and optimal conditions at which the highest phase separation yield were identified. Understanding these factors, and their interactions, is essential for optimizing complex coacervate-based formulations for infant nutrition.
The effects of spray-drying or freeze-drying on the stability and reformation of complex coacervates were also investigated. The complex coacervation of LF with β-casein increased the denaturation point of LF by 5 °C; however, this increase does not permit commercial thermal treatment of LF without anticipated denaturation and functional loss. Importantly, drying the complex coacervates and rehydrating them showed no significant impact on the rheological behavior and all complex coacervates exhibited a frequency-dependent viscoelastic behavior. Potentially, freeze-drying complexes formed under infant nutrition grade conditions and dry-blending coacervated ingredients into infant formula may avoid further thermal processing and ensure that bioactivity is retained.
The final chapters investigated the stability of complex coacervates during infant in vitro digestion and their potential impact on gastrointestinal health. Complex coacervates showed altered gastric proteolysis, which resulted in different peptide profiles, and could potentially influence bioactivity. Additionally, the digested complex coacervates were studied for their anti-inflammatory properties using a cell T84 epithelial cell model. The bifidogenic properties were studied by stimulating Bifidobacterium longum ssp. infantis with digested complex coacervates; all digested samples exhibited bifidogenic effects, with undigested β-casein stimulating bifidogenesis the most.
The findings of this research will further support the development of LF and β-casein complex coacervation as a potential way of producing novel ingredients, as their unique physicochemical properties, increased heat-stability, and altered peptide profiles upon digestion, potentially leading to different biological activities, make them interesting to consider for inclusion in infant nutrition.