Food and Nutritional Sciences - Doctoral Theses
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Item Upcycling of brewing by-products using bio-processing technology and an analysis of their functional and nutritional characteristics(University College Cork, 2024) Jaeger, Alice; Arendt, Elke K.; Sahin, AylinThis thesis focuses on the upcycling of brewing by-products; specifically brewer’s spent yeast (BSY) and a protein isolate produced from brewer’s spent grain (BSG). Although high in nutrients, the unappealing sensory characteristics and unstable nature of these products has made it difficult to valorise these materials to date. Spent yeast was extensively characterised and a process was developed focused on alcohol removal, standardisation, autolysis, and lactic acid bacteria fermentation using L. amylovorus FST 2.11. As a result of this process, sensory characteristics were significantly improved regarding reduced bitterness and an increase in fruity flavours. Techno-functional properties were also altered, with major changes in pH and protein composition. In addition, the effects of this processing on protein digestion, using the in-vitro INFOGEST protocol, and on the gut microbiome, using an in-vitro colon model were examined, yielding increased amino acid bio-availability and indicators of prebiotic potential. As well as BSY, the potential of BSG as a sustainable nutrition source was examined, particularly regarding the functional and digestive properties of a spent grain protein isolate (BRP). BRP was shown to have superior functionality to many plant proteins, in particular regarding solubility, and also displayed good digestibility characteristics as determined using the tiny-TIM in-vitro model. This research explores the enormous potential to be found within brewing by-products and the benefits of processing to achieve palatable, functional and highly nutritious food ingredients to form a part of a more sustainable and circular future food system.Item Investigating the drying kinetics, quality attributes, proteome profile and the generation of aroma compounds during dry-ageing of grass-fed beef(University College Cork, 2024) Álvarez Hernández, Sara; O'Neill, Eileen; Mullen, Anne Maria; Álvarez, Carlos; Instituto Nacional de Investigacion Agropecuaria, Uruguay; TeagascBeef quality influences the consumer’s eating experience and their purchase intentions. Eating quality is impacted by many factors, such as, genetics and production systems, slaughtering management, post-mortem interventions, meat ageing, and packaging of meat products (Álvarez, Mullen, et al., 2021). Ageing consists of a subsequent series of biochemical events that start once the rigor sets in (when the meat reaches its maximum toughness after the slaughter of the animal) and finishes once the beef is ready for consumption. During this tenderisation period, muscle proteolytic enzymes (e.g. calpains, cathepsins, caspases) break down myofibrillar and cytoskeletal proteins, contributing to the degradation of the muscle structure and the improvement of meat tenderness (Bhat et al., 2018a). This thesis focuses on the study of ageing or tenderisation phase, particularly on the process known as dry-ageing, which has been significantly investigated over the last two decades. Dry-ageing of beef consists of storing carcasses, primals or sub-primal cuts without protective packaging in a chamber or unit under controlled refrigerated air conditions (relative humidity of 70-80%, temperature of 0-4 °C, and airflow of 0.5-2.5 m/s) for 14-35 days (Koutsoumanis et al., 2023). However, restaurants and retailers may use longer ageing times than 35 days if safety of the meat is demonstrated (Point 2a of Chapter VII in Section I of Annex III, Regulation (EC) No 853/2004 amended on 14th of December 2023; See more details in Appendix I of this thesis). Dry-ageing is a value-adding process that improves meat tenderness and results in a product with unique flavour characteristics. For a long time, dry-ageing was the only method of ageing available to meat processors and was considered a craft process where each meat processor would use different ageing days and air conditions, developing unique dry-aged meat products. Following the development of new packaging and processing technologies in the 1970s, wet-ageing (where meat is vacuumed-packaged using water-impermeable films and aged) progressively replaced dry-ageing, since it overcame some of the disadvantages associated with dry-ageing (Savell, 2008), such as the higher moisture and trimming losses, longer processing times, and higher production costs (Terjung et al., 2021). There is a general agreement that dry-ageing generates tender beef with unique flavour nuances, for which niche markets are willing to pay premium (Berger et al., 2018; Sitz et al., 2006). This signature “dry-aged beef” flavour has been described as nutty, aged, beefy, and brown roasted, and obtained higher liking scores than wet-aged beef (Campbell et al., 2001; Kim et al., 2016; Li et al., 2014; Stenström et al., 2014; Warren & Kastner, 1992). The development of a dry-aged beef flavour is influenced by a number of factors, such as dehydration, endogenous proteolysis, lipid oxidation, and the enzymatic activity of microorganisms on the beef surface (Zhang et al., 2022). These factors contribute to the generation of taste- and aroma-related precursors during ageing, which can further take part in complex reactions during cooking, such as the Maillard reaction, the thermal oxidation/degradation of lipids, and the interaction of these two pathways to produce high flavour-impacting compounds (Zamora & Hidalgo, 2011;Zamora et al., 2015). In addition, dehydration during dry-ageing favours the concentration of the above-mentioned flavour-related compounds (Lee, Choe, et al., 2019) and the lower moisture content and water activity may also enhance the Maillard reaction during ageing (Gardner & Legako, 2018; Li et al., 2021). The potential of dry-ageing to produce a unique premium product has increased the interest of purveyors and retailers in dry-aged beef in recent years (Dashdorj et al., 2016). Likewise, research on dry-ageing has increased in recent years and focused on aspects such as its impact on the meat quality attributes (Berger et al., 2018), understanding the generation of flavour precursors and volatile compounds during dry-ageing (Setyabrata, Vierck, et al., 2022), as well as, the characterisation of the microbiome in dry-aged beef (Oh et al., 2019). Despite moisture evaporation being an intrinsic part of dry-ageing, research focusing on dehydration dynamics is scarce. There is also a lack of research in the proteomic changes occurring during dry-ageing and the impact of post-mortem interventions on the final product attributes. In this context, the present thesis addresses several aspects of dry-ageing in order to increase our fundamental understanding of the process with a view to facilitating improvements in the efficiency and sustainability of the process while maintaining product quality. Therefore, the main objectives of this research work are as follows: ▪ To investigate the drying dynamics during dry-ageing of grass-fed beef. The outcomes obtained from this research will help meat processors to understand how water evaporates during dry-ageing, and may assist in optimisation of the ageing time. ▪ To study the proteome evolution during dry-ageing and understand the biochemical changes that determine the development of dry-aged beef products. In addition, this project aims to identify potential biomarkers that may further help in tailoring ageing time and meat quality attributes of dry-aged beef products ▪ To assess the application of a post-mortem intervention, namely UV treatment prior dry-ageing to improve process efficiency (by influencing drying rate) while maintaining desirable quality attributes. ▪ To explore the flavour chemistry by identifying and quantifying the aroma compounds that contribute to the unique dry-aged flavour of grass-fed beef. This work also investigates the influence of UV treatment before dry-ageing on the aroma profile of dry-aged beef.Item The formation and physiochemical properties of Tremella fuciformis polysaccharide-proteins complex(University College Cork, 2024) Tian, Lili; Miao, Song; Roos, Yrjo; O'Sullivan, Maurice; China Scholarship Council; TeagascProtein-polysaccharide electrostatic interactions have received increasing research interest during the last two decades due to their potential applications in food systems. The nature of protein-polysaccharide interactions varies depending on biopolymer characteristics and environmental conditions. However, the effects of internal and external factors on protein-polysaccharide interactions and complex formation have not been thoroughly studied. This study compares the electrostatic interactions of different protein-polysaccharide systems under varying solution pH levels, biopolymer concentrations, and biopolymer ratios. Additionally, the impact of physical treatments on these interactions and the resulting physicochemical properties of the protein-polysaccharide complexes are investigated. Tremella fuciformis polysaccharide (TPS) was extracted and characterized for its chemical composition and functional properties. As an anionic polysaccharide, TPS exhibited a high viscosity and strong emulsion-stabilizing ability. The interaction between TPS and whey protein isolate (WPI) at a biopolymer concentration of 0.1% was strongest at pH 3.2 with a WPI/TPS ratio of 2:1. Compared to gum Arabic (GA), TPS showed a stronger affinity to WPI with more binding sites. When WPI was compared to lentil protein isolate (LPI) and soy protein isolate (SPI), both similarities and differences were observed in their complex coacervation with TPS and in the characteristics of emulsions stabilized by these protein-TPS complexes. The turbidity curves showed that the onset pH of soluble complex formation remained unchanged for all protein-TPS systems, and the onset pH of insoluble complex formation shifted to higher values with increasing protein/TPS ratios; the pH at which the maximum amount of insoluble WPI:TPS and SPI:TPS complexes formed shifted to higher values with increasing protein/TPS ratios, a shift that was not observed for the LPI:TPS system, likely due to the lower solubility of LPI. The main components of each protein interacting with TPS have been identified. Different proteins exhibited distinct changes in secondary structure when interacting with TPS. At pH 4.5, a high ratio of TPS increased the stability of protein emulsion, regardless of protein type. However, WPI:TPS emulsions with low TPS ratios were extremely destabilized due to the formation of insoluble WPI:TPS complex coacervates, a phenomenon not observed in the emulsions stabilized by LPI:TPS and SPI:TPS groups. To address LPI’s low solubility, the ultrasound treatment (UT) was applied. The interaction between sonicated LPI and TPS was studied. As the duration of sonication increased, the sonicated LPI samples exhibited decreased intrinsic fluorescence, higher zeta potential values, and increased solubility. The pH at which the maximum amount of insoluble LPI:TPS complexes formed shifted to higher values as the LPI solubility improved. The longest ultrasound treatment (40 min) resulted in the strongest binding affinity of LPI with TPS, corresponding with the highest viscosity observed in their mixture solution as the amount of complex formation increased. The effect of UT on the physicochemical properties of three WPI:TPS mixture systems at pH 3.0, pH 4.5, and pH 6.0 was studied. The electrostatic interaction between WPI and TPS at pH 4.5 and 6.5 could be improved by appropriate UT (20 min), as revealed by the higher turbidity and decreased absolute zeta-potential value of their mixture solution. Increases in β-sheet and random coil were observed for WPIs and WPI:TPS systems sonicated for 20 min. The surface hydrophobicity of the WPI:TPS system at pH 4.5 increased significantly with the duration of UT; moreover, this system showed enhanced emulsifying properties following appropriate UT. On the whole, this thesis will provide important information on the impact of external factors, ultrasound, and protein types on the interaction of TPS with proteins, which may lead to the development and application of new protein-polysaccharide-based food ingredients or structured products.Item 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; NestléMilk 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.Item 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 NestléLactoferrin (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.