Food and Nutritional Sciences - Doctoral Theses
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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.Item The role of micronutrients and impact of fortified foods in the diets of teenagers in Ireland(University College Cork, 2024) Walsh, Niamh M.; Walton, Janette; Kehoe, Laura; Flynn, Albert; Cashman, Kevin; Coffey, Aidan; Department of Agriculture, Food and the Marine, IrelandDuring the teenage years, adequate micronutrient intake is crucial for physical and cognitive development. Strategies to meet high nutritional needs can involve food based dietary guidelines (FBDG) or fortification of foods and recommendations for nutritional supplements. The aims of this thesis were to estimate current micronutrient intakes in the diets of teenagers in Ireland, investigate the dietary determinants of vitamin D and calcium and examine the role of fortified foods in teenagers. The analyses for this thesis were based on data from the National Teens’ Food Survey II (NTFS II), a nationally representative study which collected data on food and nutrient intakes of 428 teenagers (13-18y) in the Republic of Ireland between 2019-2020. Dietary data were collected using weighed food diaries and nutrient intakes were estimated using UK and Irish food composition data. Mean daily intakes (MDI) and usual intakes of food groups and nutrients were estimated using SPSS©. The prevalence of inadequate and excess intakes were estimated using estimated average requirements (EAR) and the tolerable upper intake level (UL). Dietary determinants of vitamin D and calcium intake were investigated by examining the food groups and consumption patterns which contributed to the difference in intakes between the high and low intake groups. A fortified food consumer was defined as someone who consumed fortified foods at least once during the recording period. A large proportion of teenagers in Ireland had inadequate intakes of micronutrients, particularly vitamin D (94%), calcium (51%), vitamin C (48%), and zinc (48%). There was little risk of excessive intakes with few exceeding the UL for folic acid (4%), copper (<1%), and zinc (<1%). The key sources of micronutrients included natural animal and plant sources and fortified foods, with supplements contributing to 0-6% of intakes. Considering the dietary determinants of vitamin D intake, 100% in the low intake group had intakesItem The role of dietary fibre and plant-based foods in the diets of teenagers (13-18y) in Ireland(University College Cork, 2024) McGowan, Clíodhna; Walton, Janette; Kehoe, Laura; Flynn, Albert; Cashman, Kevin; Coffey, Aidan; Department of Agriculture, Food and the Marine, IrelandBackground: Adequate nutrition during the teenage years is essential to support optimal growth and lay the foundations for long term health. This life stage is also characterised by social development, increasing autonomy and independence and is a critical stage for the optimisation of health and health-related behaviours as habits developed during this time often continue into adulthood. Dietary fibre is a nutrient of interest in public health due to its essential role in normal laxation and association with reduced incidence of non-communicable diseases. Plant-based foods such as ‘fruit & vegetables’ and ‘cereals, grains & potatoes’ are key sources of dietary fibre in the diet, and feature prominently in all food based dietary guidelines (FBDG) due to their nutrient density and association with a number of health benefits. Data from the National Teens’ Food Survey (NTFS) (2005-06) (a nationally representative survey of teenagers in Ireland) reported an intake of dietary fibre below recommendations, and a poor compliance with the Irish FBDG for ‘fruit & vegetables’ and ‘cereals, grains & potatoes’. There have been many changes in the environmental, social and cultural contexts which influence teenagers lifestyles including dietary choices since the NTFS, along with updated international recommendations for dietary fibre intake for teenagers and updated Irish FBDG. New detailed dietary data for teenagers has become available from the nationally representative National Teens’ Food Survey II (NTFS II) (2019-20). Aim: The overall aim of this PhD thesis was to use data from the NTFS II (2019-20) to examine intakes of dietary fibre and the role of plant-based foods such as ‘fruit & vegetables’ and ‘cereals, grains & potatoes’ in the diets of teenagers (13-18y) in Ireland. An additional aim of this thesis was to examine changes over time compared to the previous NTFS (2005-06). Methods: The analyses for this thesis were based on data from the NTFS II (13-18 years, n 428) and the NTFS (13-17 years, n 441). Dietary intake data was collected at brand level using a 4-day weighed food record (NTFS II) and a 7-day semi-weighed food record (NTFS). Energy and nutrient intakes were estimated using Nutritics© (NTFS II) and WISP© (NTFS) based on food composition data from McCance & Widdowson’s ‘The Composition of Foods’ 5th, 6th and 7th editions (plus all nine supplemental volumes). During both surveys, modifications were made to the food composition database to include recipes of composite dishes, fortified foods, generic Irish foods and new foods on the market. Usual intakes of energy and dietary fibre were estimated using the National Cancer Institute (NCI) method using SAS Enterprise Guide©. Dietary fibre intakes were compared to the adequate intake (AI) for normal laxation, proposed by EFSA for this age group (13-14y: 19g/d, 15-17y: 21g/d, 18y: 25g/d). All further analyses were conducted using SPSS© Version 29.0. The percent contribution of food groups to the mean daily intake (MDI) of dietary fibre were calculated by the mean proportion method. Differences in the intake of dietary fibre between sexes (boy, girl), age groups (13-14y, 15-18y) and surveys (NTFS II, NTFS) were calculated using independent sample t-tests. Dietary determinants of dietary fibre intake were investigated by splitting the population into low, medium and high dietary fibre intake groups and examining the key food groups which contributed to the difference in dietary fibre intake between the high and low intake groups and associated patterns of consumption of these food groups. Differences in the MDI of dietary fibre between the low, medium and high intake groups were assessed using ANOVA and Tuckey’s post-hoc analysis. The difference in the percentage of consumers of each food group identified as determinants of dietary fibre intake between dietary fibre intake groups (low, high) were assessed using the Chi-Square test for independence. The differences in consumption patterns between the low and high dietary fibre intake groups were assessed in consumers only using the Mann Whitney U test. To examine the role of ‘fruit & vegetables’ and ‘cereals, grains & potatoes’ in the diets of teenagers in Ireland, the MDI of each food group and subgroup was estimated by summing the weight of each food group consumed for each participant and dividing the total by the number of recording days (NTFS II: 4, NTFS: 7). The percent contribution of ‘fruit & vegetables’ and ‘cereals, grains & potatoes’ to energy, macro- and micro-nutrient intakes were calculated by the mean proportion method. Differences in the intake of ‘fruit & vegetables’ and ‘cereals, grains & potatoes’ between sexes (boy, girl), age groups (13-14y, 15-18y) and surveys (NTFS II, NTFS) were calculated using independent sample t-tests. Analyses to estimate intakes of fruit & vegetables included intake of fruit and/or vegetables from composite dishes and the calculation of inedible or unconsumed portions. To assess compliance with Irish FBDG, intakes of ‘fruit & vegetables’ and ‘cereals, grains & potatoes’ were converted to servings using the Irish FBDG recommendations and standard food portion sizes. Results: Teenagers in Ireland had a usual intake of dietary fibre of 16.9g/d, which was below the AI proposed by EFSA across all age groups examined (13-14y, 15-17y, 18y). Teenage boys had higher usual intakes of dietary fibre compared to girls, however, girls had higher energy adjusted intakes, indicating girls consume a more fibre dense diet. Older teenagers (18y) had higher absolute and energy adjusted intakes of dietary fibre than younger teenagers (13-14y, 15-17y), indicating that older teenagers consumed a more fibre dense diet. The key sources of dietary fibre were identified as ‘bread & rolls’ (19%) (‘white bread & rolls’, 10%), ‘grains, rice, pasta & savouries’ (14%), ‘potato & potato products’ (13%) (‘chipped, fried & roasted potatoes’, 8%), ‘breakfast cereals’ (12%) (RTEBC, 11%), ‘meat & meat products including dishes’ (9%), ‘vegetables & vegetable dishes’ (9%) and ‘fruit & fruit juices’ (7%). Intakes of dietary fibre have increased in teenagers in Ireland since the NTFS (2005-06) (16.9g/d vs 15.5g/d), albeit remain below the EFSA AI for all age groups examined. The increased intakes are due to a higher intake of dietary fibre from ‘grains, rice, pasta & savouries’ and ‘breakfast cereals’ in teenagers in the NTFS II compared to the NTFS. To identify the dietary determinants of dietary fibre intake, teenagers were split into low, medium and high dietary fibre intake groups based on their MDI of dietary fibre. Those in the high intake group had over double the intake of dietary fibre compared to those in the low intake group (24g/d vs 11g/d). The main food groups that contributed to the difference in dietary fibre intake between the high and the low intake groups were ‘high fibre RTEBC’ (17%), ‘wholemeal & brown bread & rolls’ (11%), ‘vegetables & vegetable dishes’ (14%), ‘grains, rice, pasta & savouries’ (10%) and ‘fruit’ (10%). For ‘high fibre RTEBC’ there were more consumers in the high intake group compared to the low intake group (53% vs 33%) and those in the high intake group consumed it more often over the 4 days (2.8 vs 2.0 times) and consumed more at each eating occasion (72g vs 47g). For ‘wholemeal & brown bread & rolls’ there were more consumers in the high intake group compared to the low intake group (63% vs 24%). For ‘grains, rice, pasta & savouries’ there was no difference in the number of consumers between the groups but those in the high intake group consumed it more often over the 4 days (3.2 vs 2.4 times). For ‘vegetables & vegetable dishes’ and ‘fruit’, both food groups were more likely to be consumed by teenagers in the high intake group compared to the low intake group (vegetables & vegetable dishes: 90% vs 75%, fruit: 89% vs 57%) and be consumed more frequently during the recording period (vegetables & vegetable dishes: 6.9 vs 3.4 times, fruit: 5.5 vs 2.4 times). ‘Fruit & vegetables’ were consumed by all teenagers in the NTFS II with an MDI of 218g/d (‘fruit & fruit juices’: 122g/d and ‘vegetables’: 96g/d). Teenagers in the NTFS II had lower intakes of fruit & vegetables compared to teenagers in the NTFS (NTFS II: 218g/d, NTFS: 240g/d). The MDI of 218g/d equated to approx. 2.7 servings of ‘fruit & vegetables’ per day. This is below the Irish FBDG recommendation of 5-7 servings/day, with the majority of teenagers (92%) not meeting the recommended 5-7 servings per day and 22% of teenagers consuming less than one serving (80g) of ‘fruit & vegetables’ per day. For ‘vegetables’, 50% of intakes were from ‘vegetables in composite dishes’, which highlights the important contribution of composite dishes to vegetable intakes in teenagers, and the importance of including fruit and vegetables in composite dishes in estimates of fruit and vegetable intake (particularly for vegetables). ‘Fruit & vegetables’ contributed a small proportion of energy (5%) in the diets of teenagers in Ireland and made important contributions to the intake of dietary fibre (16%) and several micronutrients which have been identified as low in this population, including vitamin C (42%), vitamin A (24%), potassium (14%), folate (DFE) (14%) and vitamin B6 (11%). ‘Fruit & vegetables’ also contributed to the intake of total sugars (20%), due to the natural sugars present in ‘fruit & fruit juices’ and a negligible amount to free sugars (2%) due to ‘fruit juices & smoothies’. ‘Cereals, grains & potatoes’ were consumed by all teenagers in the NTFS II with an MDI of 345g/d (‘grains, rice, pasta & savouries’:128g/d, ‘total bread’: 85g/d, ‘potato & potato products’:84g/d and ‘breakfast cereals’: 48g/d). The overall food group contributed 4.8 servings for boys and 3.8 servings for girls with 1.6 and 1.1 servings, respectively, from ‘wholemeal & brown breads, cereals, pasta and rice and unprocessed potatoes’. This is well below the recommended intake of 4-7 and 3-4 servings per day for boys and girls respectively of ‘wholemeal & brown breads, cereals, pasta and rice and unprocessed potatoes’ in the Irish FBDG. ‘Cereals, grains & potatoes’ contributed significantly to intakes of energy (39%), carbohydrate (55%), protein (30%) and dietary fibre (60%) and made important contributions to a number of micronutrients including B vitamins (15-50%), vitamin D (25%), vitamin C (21%), vitamin E (22%), calcium (35%), potassium (33%) and iron (22%) in the diets of teenagers in Ireland. ‘Cereals, grains & potatoes’ also contributed to intakes of saturated fat, sodium and free sugars, primarily due to specific subgroups including ‘pizza’, ‘savoury dishes & products’ and ‘chipped, fried & roasted potatoes’, ‘bread’ and ‘breakfast cereals’. Teenagers in the NTFS II had lower overall intakes of ‘cereals, grains & potatoes’ compared to teenagers in the previous NTFS (NTFS II: 345g/d, NTFS: 364g/d) with lower intakes of ‘potatoes & potato products’ and ‘low fibre RTEBC’ and higher intakes of ‘grains, rice, pasta & savouries’. Conclusion: In conclusion, the findings from this thesis indicate that overall, teenagers in Ireland are not meeting dietary guidelines for dietary fibre, fruit & vegetables and cereals, grains & potatoes. However, a positive finding was that there is a proportion of the teenage population that had intakes of dietary fibre that reached the AI proposed by EFSA based on existing dietary patterns, providing an evidence base to develop dietary strategies to improve dietary fibre intake in this population group. Based on this evidence, these strategies should focus on increasing intake of ‘fruit & vegetables’ and promoting a swap from refined grains to wholemeal & brown breads, cereals, pasta and rice and unprocessed potatoes which would improve intakes of dietary fibre in teenagers in Ireland and also support FBDG for ‘fruit & vegetables’ and ‘cereals, grains & potatoes’, which have a much wider nutritional role in the diet as evidenced in this thesis. Findings from this thesis may provide evidence to public health campaigns to improve dietary fibre intakes and promote the consumption of plant-based foods to increase compliance with the Irish FBDG in teenagers in Ireland. The findings from this thesis may also provide information to policy makers across various sectors of Irish society including education and health to gain a more comprehensive understanding of the plant-based foods influencing intakes of dietary fibre in teenagers in Ireland.Item Plant protein structure design and its potential substitution of dairy proteins in gelation systems(University College Cork, 2023) Tang, Qi; Miao, Song; Roos, Yrjo; O'Sullivan, Maurice; China Scholarship Council; Horizon 2020Plant-based proteins are gain attention as versatile alternatives to dairy-derived proteins as functional ingredients in food formulations due to their greater sustainability and lower production costs, and health benefits. However, in-depth knowledge about the structural and functional knowledge of plant and dairy proteins under different pH conditions and the modifying technologies for binary plant and dairy proteins have rarely reported. The study investigated the pH-dependent behavior of structural, and functional properties, especially thermal gelation performance, of plant protein and dairy proteins. Moreover, the study also explored the modifying strategies on the gelation performance of lentil-dairy binary protein gels treated with by heating and microbial transglutaminase. The studies focused on the influence of different pH levels on the structural, surface properties and functional properties of different plant proteins and dairy proteins. Findings demonstrated that dairy proteins generally showed brighter color (higher L* and lower a* and b*), higher solubility, emulsification properties, and foaming capacity compared to plant proteins, whereas plant proteins displayed superior foaming stability and water holding capacity. The thermal gelation performance of plant proteins and dairy proteins are also showed that the disulfide bond formation was attributed to partial 11S acidic and basic subunits of plant proteins (soy, pea, lentil, and chickpea). The disulfide-bonded aggregation was attribute to partial β-Lg and α-La in WPI, which was inhibited at acidic conditions but facilized under higher pH conditions, whereas in CM, κ-casein and αs2-casein might involve in this process. Moreover, the highest gel performance of SPI and CPI were peaked at pH 9, but that of LPI at pH 3, whereas no self-standing gel formed for PPI at the same protein concentration (14%, w/w) over the test pH conditions. For dairy protein, whey protein showed the superior gel performance at pH 7, whereas that was observed optimum at pH 3 for casein micelles. The modification strategies were further explored by subjecting plant and dairy binary protein mixtures to heating and microbial transglutaminase treatment and demonstrated that only certain fractions (legumin acidic and basic subunits) in LPI involved in disulfide-mediated polymerization, whereas almost all fractions in WPI were involved, but that of CM was hindered during heating treatment. However, the 11S acidic subunit and 7S vicilins in LPI were involved in Gln-Lys isopeptide bond formation when subjected to MTGase treatment, whereas that was hindered in WPI but promoted in CM. The binary protein gel performance demonstrated that the application of heating treatment could result in the formation of self-standing gels when replacing whey protein with lentil protein. Nevertheless, when aiming to substitute casein micelles with lentil protein with good gel performance, microbial transglutaminase treatment was found to be a more effective modifying method. Furthermore, a higher ratio of casein micelles to lentil protein can lead to a significant enhancement in gel performance, resulting in improved mechanical properties, rheological properties, water-holding capacity, and the development of more homogeneous and compact microstructures. Remarkably, when substituting 25% casein with lentil protein, similar gel performance with casein alone was observed when mediated by microbial transglutaminase treatment. The findings of this study offer a profound comprehension of the distinctions between plant proteins and dairy proteins regarding their structural and functional properties under varying pH conditions. Furthermore, this research provides different modifying strategies for substituting dairy proteins with plant proteins, thereby broadening the potential applications of plant proteins in diverse food formulations.