Food and Nutritional Sciences - Doctoral Theses
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Item The impact of genetic variation on functional, technological and digestive properties of β-lactoglobulin(University College Cork, 2025) Kelly, Liam M.; O'Mahony, Seamus Anthony; Tobin, John; Science Foundation Ireland; Department of Agriculture, Food and Marine; TeagascGenetic variations of milk proteins have been studied since their discovery in 1946. Since then, over 50 variants have been found among the four caseins (αs1-, αs2-, β-, and κ-casein) and the two main whey proteins, β-lactoglobulin (β-lg) and α-lactalbumin. In the early 1990’s, it was speculated that certain genetic variants of β-casein could be associated with negative health effects, including heart disease, diabetes and schizophrenia. While no scientific evidence has since been published in support of these health claims, these reports did stimulate a renewed scientific and commercial interest in genetic variants of milk proteins, with some companies even structuring their business models around them, such as the a2 Milk Company (Auckland, New Zealand). Undoubtedly, there is potential for genetic variation among the milk proteins to alter functional, technological and possibly nutritional and digestive properties of dairy products. Thus, research is required to define potential beneficial and deleterious effects of specific protein variants on the processing and nutritional quality of milk. Irish herds generally do not select for specific genetic variants as part of the national breeding programme, resulting in a diverse pool of variants among Irish cows. Initially, to support understanding of the distribution of genetic variants within an Irish herd, the protein profiles of 48 cows was assessed; of these cows, 17 genetic variants were identified among β-casein, κ-casein and β-lg. The A2A2 β-casein genotype had the highest frequency in Holstein Friesian (HF) cows; however, the A1I and the A2B genotypes were associated with higher levels of true protein and casein. In addition, it has also been found that milk with the A variant of β-lg was associated with higher levels of total whey proteins compared to milk with the B variant. This difference in β-lg concentration has been linked to the DNA mutations that cause the genetic variation being located close to the promoter region for gene expression. The differences in the macro composition of milk as a result of genetic variation could be of benefit to both the farmer and milk processor, in terms of farm gate milk price and differentiated added value products, respectively. Additionally there may also be a benefit to consumers who seek milk products with designated milk genetic variants. Identification of the genetic variants among 48 cows, using high performance liquid chromatography analysis, indicated that the AA, AB and BB genotypes of β-lg were commonly distributed among the cows tested. As β-lg is the principal whey protein on a mass basis, it determines the nutritional and functional quality of subsequent whey protein ingredients, such as concentrates and isolates. These whey protein ingredients, produced from pre-treated cheese whey, or isolated directly from milk, through a combination of microfiltration (MF), ultrafiltration (UF)/nanofiltration and spray drying, often find application in high value, life-stage nutritional products. Thus, the primary whey protein β-lg, its genetic variants and ingredients/products produced therefrom, are the primary research focus of this thesis. The primary structural differences between the A and B variants of β-lg are due to amino acid substitutions that occur at positions 64 and 118. At position 64, the A variant has an aspartic acid, whereas the B variant has a glycine. Aspartic acid contains a charged functional group, while the glycine functional group is not charged. This substitution in particular causes the A variant of β-lg to have a greater net negative charge. The substitution that occurs at position 118, in which the A variant has a valine and the B variant has an alanine is not thought to significantly impact the structure, and thus functionality, of β-lg. Most studies to date have assessed the functional properties of β-lg in milk, a complex system in which other components may present confounding factors which mask differences in functionality attributable to the protein variant. By purifying the whey protein fraction of milk into a whey protein isolate (WPI) containing either the A or B variant of β-lg, the true effects of genetic variation can be assessed with limited interference from other components of milk. As this thesis had already identified cows that produced only the homozygous AA or BB genotype of β-lg, their milk was segregated for separation of the individual β-lg variants using a combination of MF and UF. MF is widely used in the manufacture of WPIs from pre-treated cheese whey, and also in the separation of caseins and whey proteins from skim milk. While separation and purification of the β-lg variants was the primary goal, their impact on the efficiency of whey protein separation was also assessed. The results of this work demonstrated that the accumulation of reversible and irreversible foulants, rejection of whey proteins and overall process efficiency were determined by the β-lg variant present in the skim milk. This is not surprising, considering that fouling of the membrane plays a critical role in both flux decline, and by proxy, process efficiency, with β-lg reported to be a major contributor to fouling. It was found that transmission of whey proteins during MF of skim milk was higher for the skim milk containing the A variant than the B variant of β-lg, and while the skim milk containing the A variant had higher accumulation of both total and reversible fouling than the skim milk with the B variant, it was not rate limiting relative to whey protein permeation. These results indicate that skim milk with the A variant of β-lg could be advantageous to whey processors in terms of process efficiency and yield. Analysis of the WPIs produced by a combination of MF and UF described in this thesis identified unique functional properties specific to the β-lg variants. The thermal denaturation and gelation onset temperatures were higher for the WPI with the B variant of β-lg. However, despite the A variant having a lower denaturation/gelation temperature, it had better stability against heat-induced gel formation in the pH range 6.6-7.2. These differences in functionality, specifically in terms of denaturation and aggregation processes, presented additional research questions surrounding behaviour of the proteins in more complex products. By manufacturing WPI containing either β-lg A or B genetic variants, and observing unique functional properties between the variants, the research question was proposed as to whether the differences between the variants would also be evident in a more complex dairy product, and whether the variants had a significant effect on in vitro digestion behaviour. Infant milk formula (IMF) was thus chosen as a suitable system to evaluate the β-lg variants, as it constitutes approximately 50% of the protein in a 0-6 month formula. By creating IMF with different heat treatments of either high temperature short time (HTST) or ultra-high temperature (UHT), containing either the A or B variants of β-lg, the impact of the genetic variation of β-lg on digestion was assessed using a semi-dynamic infant digestion model. While the effect of heat treatment between the samples was very clear, surprisingly, there was little difference between the β-lg variants during simulated digestion, in terms of curd formation, protein breakdown and free amine concentrations at the end of digestion. Overall, the original research presented in this thesis provides new and important insights into the impact of genetic variation on the functional, technological and digestive properties of β-lg, by producing purified whey ingredients for evaluation at semi-commercial scale. Overall, there does appear to be a case for selection of the A variant of β-lg in the dairy herd, especially for dairy processors who specialize in whey protein ingredients.Item Influence of formulation and processing strategies on the physicochemical properties of lentil protein stabilised emulsions for use in infant nutritional applications(University College Cork, 2024) Malterre, Nicolas; O'Mahony, Seamus Anthony; Arendt, Elke K.; Francesca, Bot; Horizon 2020The interest and research for plant-based infant nutritional products is growing together with the attention for a sustainable food system. However, while the formulation of plant-based food products is widely studied, the investigations of processing innovations for the manufacture of sustainable, nutritional plant-based food systems are more limited. This thesis investigates the effect of processing techniques to formulate lentil protein-stabilised emulsions at high total solids for young child nutritional products, with enhanced techno-functional properties and physical stability. Formulation of high-solid lentil protein emulsions (lentil protein, sunflower oil and maltodextrin representing 15.85, 27.43 and 56.72% of total solids, respectively), showed high physical and heat stability in the range between 23 and 26% total solids. On the other hand, emulsions at higher total solid concentrations (i.e., 29% and above) had poor physical stability and showed extensive oil-droplet flocculation. The low protein solubility in the high total solid emulsions was identified as one of the main limiting factors in the techno-functional properties of the emulsified systems. Therefore, different physical pre-treatments, including high-shear mixing and high-pressure homogenisation (HPH), were selected to improve the techno-functional properties of the protein ingredients and the emulsions made therefrom. High-shear mixing showed an increase in protein solubility (as measured through nitrogen content in the soluble fraction) (from 46.87 to 68.42% after 0 min and 15 min at 15,000 rpm, respectively), and overall enhanced physical stability of the dispersions, with a reduction of the separation rate from 71.23 (0 min) to 24.16%·h-1 (15 min). HPH allowed an enhanced solubility compared to the high-shear mixing process and at 150 MPa the lentil protein isolate dispersions had a solubility of 96.4%. Moreover, the pre-treated lentil protein dispersions (5%, w/v, homogenised at 150 MPa), had higher hydrophobicity (from 607 ± 29 to 1271 ± 50), as well as smaller particle size (from 10.7 to 0.27 μm) and higher physical stability (from 8.12 to 4.97 %·h-1) compared to untreated samples. To further understand the impact of the pre-treatments on the techno-functional properties of lentil protein emulsions, HPH was selected as pre-treatment due to its high performance compared to high shear mixing and the effects of 0, 15, 50 and 150 MPa on the techno-functional properties of lentil protein dispersions and on the emulsions made therefrom were investigated. The results showed that increasing the solubility of the protein ingredient by HPH pre-treatment led to a significant improvement of the techno-functional properties of the subsequent emulsions. Particularly, emulsions formulated with HPH pre-treated lentil protein ingredient at 50 MPa displayed small oil globules (from 1.40 to 1.19 μm), enhanced heat stability with less flocculation and overall higher physical stability compared to the untreated sample (from 16.75 to 2.05%·h-1). Furthermore, to better understand the colloidal stability of the emulsions at the processing conditions generally used in infant formula manufacture, the effect of combined HPH (50 MPa) and heat-treatment (120°C for 60 s) was investigated. A lentil protein dispersion subjected to HPH (50 MPa) and heat (120°C for 60 s) treatment was produced, which enabled the subsequent formulation of a high total solids (29% w/v) lentil protein-stabilised emulsion, with enhanced techno-functional properties as compared to the untreated sample, such as lower viscosity (from 19.18 to 7.43 mPa·s) and reduced flocculation, with high physical stability (from 15.11 to 1.85 %·h-1). These improvements in the physico-chemical properties can be linked to higher protein solubility, as well as reduced particle size of the protein dispersion, induced by HPH and heat treatment. Furthermore, these results highlighted that combined HPH and heat treatment can support the development of physically stable emulsions at high total solids. The novel scientific findings presented in this thesis contribute to a better understanding of the impact of processing techniques and subsequent improvement of protein solubility, for enhancing the techno-functional properties of plant protein-stabilised emulsions for use in the manufacture of sustainable plant-based young child infant formula.Item Investigating casein-polymer interactions as a platform technology for cheese for Asian consumers(University College Cork, 2024) Ouyang, Hao; Kelly, Alan; Sheehan, Diarmuid (JJ); Dairy Research Ireland; TeagascThe focus of the global cheese industry on accessing new markets for cheese is driving a greater need for innovation in cheese products. Increasing opportunities for cheese have been identified in many Asian countries, due to factors such as rising income and a change away from traditional lifestyles (e.g., appeal of fast-food chains to young people). The objectives of the work presented in this thesis were: to gain a better understanding of consumers in emerging markets; to identify texture or flavour characteristics of cheese that may be preferred by such consumers; and to explore the potential of incorporation of different non-dairy polymers in different processing steps of cheese manufacturing to achieve such sensory properties. Consumer focus group methodology was applied to understand the preference for, and consumer behaviour towards, cheese products by a cohort of young, internationally mobile Chinese consumers. Participants in the focus groups had general positive expectations towards cheese due to associations with western-style foods and nostalgia; however, many participants had low perceived ability to select cheese and had limited motivation to engage with cheese due to low perceived relevance of cheese to their daily food life. Innovative cheese products such as cheese snacks or cheese energy bars may be developed to better fulfil important choice motives. Participants generally preferred cheese with a mild and milky flavour and a soft texture, which agreed with trends emerged from a parallel literature review. The effects of addition of konjac glucomannan (KG, neutral charge), soy soluble polysaccharides (SP, negatively charged), or chitosan (CH, positively charged) on rennet coagulation and rennet-induced casein gels were investigated to assess the potential of incorporating these polysaccharides prior to rennet coagulation to modify cheese texture. Addition of either KG (0.02%, 0.04%) or SP (0.25%, 0.5%) enhanced rennet coagulation properties by reducing rennet coagulation time (RCT) and promoting development of gel firmness. The addition of CH, on the other hand, hindered rennet coagulation by decreasing the maximum gel firming rate. The two non-interactive polysaccharides (KG and SP) affected rennet coagulation by a depletion flocculation mechanism, and hydrated KG or SP particles may act as inactive filler particles in rennet-induced gels. Larger hydrated KG particles provided weak points in the gels, while smaller SP particles did not significantly affect microstructure or properties of rennet-induced casein gels at the concentrations studied, which, combined with relatively low retention rate in curd, limits its use for modification of cheese texture. Interactions of CH with caseins hindered aggregation of hydrolysed caseins, and gave less interconnected gel structures. The incorporation of KG or CH affected rennet coagulation and gel structure through different mechanisms, but could both result in a casein matrix that is softer and more easily broken down, which may be harnessed to develop cheese preferred by Asian consumers. The effects of addition of SP/KG/CH (0.5%) at dry salting of Cheddar-style cheese manufacture on characteristics of the cheeses were examined. Addition of KG at dry salting resulted in formation of a KG gel at milled curd junctions, retaining significantly (p<0.05) higher levels of moisture and salt, and higher salt-in-moisture (S/M) level compared to the control without polysaccharide addition. A significant (P<0.05) reduction in salty whey level was also observed with addition of CH, although to a lesser extent compared to KG addition, with a concurrent significant (p<0.05) increase in salt and S/M levels. The changes in compositions of these cheeses also impacted texture and volatile compound profiles over a ripening period of 90 d, although no difference in overall consumer acceptability was observed by a cohort of Chinese consumers. Dry salting is thus a promising step to incorporate different polysaccharides into Cheddar-style cheese, which could also help with salt retention and reduction in salty whey production, as well as modifying cheese characteristics (e.g., composition and texture), which may be of interest to manufacturers and researchers from sustainability and/or product development perspective. Cheese puffs (manufactured using microwave vacuum drying: MVD) produced from Cheddar curds with or without polysaccharide addition at dry salting were characterised. MVD was shown to be a promising technology to produce dried cheese snacks with minimal changes in properties such as colour and volatile compounds. The volumetric expansion during puffing process was significantly (p<0.05) decreased by addition of KG, and increased by addition of CH, respectively; this showed that polysaccharides may be used in modifying texture of cheese puffs. Overall, the work undertaken provides a technological platform for industry to help access Asian markets and contributes to the understanding of use of polysaccharides in development of novel cheese products.Item Impact of a transition to diets from sustainable sources on micronutrient intakes and status(University College Cork, 2024) Leonard, Ursula; Kiely, Mairead; Arranz, Elena; Department of Agriculture, Food and the Marine, Ireland; Food Institutional Research MeasureBackground: A global transition to diets from sustainable sources is required for planetary health. This transition will result in greater reliance on plant-based protein sources coupled with reductions in animal source foods. The evidence base underpinning the formulation of nutrition policy lacks data on the feasibility, safety and effectiveness of translating sustainable dietary guidelines into practice in the population, which could pose risks to nutrition for large sectors, including females of reproductive age and children. Objective: The aim of this thesis was to explore of the impact of a transition to diets from sustainable sources on micronutrient (MN) intakes and status. Objectives were to systematically review the existing evidence assessing the impact of following environmentally protective diets on MN intakes and status (Chapter 2); design and conduct a first-of-its-kind dietary intervention trial of an environmentally protective diet (Chapter 3); assess the impact of the intervention on MN intakes (Chapter 4) and status (Chapter 5); and explore the potential for dietary modelling to assist the development of MN adequate diets from sustainable sources (Chapter 6). Methods: PRISMA guidelines were followed to conduct a systematic literature review (SLR) of studies reporting MN and environmental outcomes, searching seven databases from January 2011 to October 2022 (Chapter 2). Chapters 3 to 5 describe a 12-week, three-centre, single-blind, parallel, randomised controlled trial (RCT) among 355 healthy participants aged 18-64 years, known as MyPlanetDiet. The intervention provided participants with personalised dietary advice based on their dietary habits, in line with sustainable healthy principles (intervention arm) or dietary guidance based on the Irish and Northern Irish healthy eating guidelines (control arm). Outcomes included changes in dietary environmental impacts, energy, protein, MN intakes, the prevalence of inadequate intakes and changes in MN status throughout the intervention. Chapter 6 describes a narrative review of diet optimisation studies that design diets considering nutritional, environmental and context-specific constraints. Results: The SLR (Leonard et al., 2024) included 56 studies; one RCT which provided the only biomarker data; 10 dietary intake studies, and 45 dietary modelling studies, including 29 diet optimisation studies. Most studies suggested that intakes of zinc, calcium, iodine, and vitamins A, B12 and D would decrease, and total iron and folate would increase in a dietary transition to reduce environmental impacts, with similar findings for the prevalence of inadequate intakes, reported in only 10 studies. In the MyPlanetDiet RCT, dietary GHGE decreased from baseline to endpoint in both arms and was lower in the intervention arm at endpoint. There were changes in MN intakes in both arms over the 12-weeks, and at endpoint, the intervention arm had lower intakes of energy, protein, thiamin, riboflavin, niacin, vitamins D, B6, B12 and C, calcium, zinc, potassium, selenium and iodine, and higher intakes of vitamins A, E and K1 (P < 0.05) compared with controls. The prevalence of inadequate intakes of riboflavin, calcium, zinc, vitamins B6, B12 and C was higher, and of vitamin E and copper was lower (P < 0.05) among females in the intervention arm than controls. Among males, the prevalence of inadequate intakes of riboflavin, selenium and iodine was higher in the intervention arm than controls (P < 0.05). Changes in biomarkers of nutritional status analysed for this thesis were minimal over the 12 weeks. Our narrative review of diet optimisation studies (Leonard & Kiely, 2024) identified a range of diets that meet individual MN requirements, have reduced environmental impacts, and minimise deviation from culturally acceptable dietary practices. Although these are theoretical solutions, opportunities arising from the results of diet optimisation studies include supporting the development of food-based dietary guidelines and exploring food-based strategies to increase nutrient supply, such as fortification. Conclusions: The findings from our SLR, mainly based on observational data, and the MyPlanetDiet RCT aligned, identifying several key MNs that are at risk in the transition to diets from sustainable sources, particularly among females. Greater emphasis on meeting diverse MN requirements from appropriate dietary sources is required in the global discussion around developing diets from sustainable sources. Dietary modelling techniques, such as diet optimisation, can be a useful tool in the generation of hypotheses and diet solutions for testing in further intervention studies among diverse populations.Item Reconstruction of milk fat globules/membrane (MFGs/MFGM) and characterization of its physicochemical properties and techno-functionalities(University College Cork, 2024) Sun, Yanjun; Miao, Song; Roos, Yrjo; O'Sullivan, Maurice; Teagasc; China Scholarship Council; State Key Laboratory of Dairy BiotechnologyMost commercially available infant formulas (IFs) lack phospholipids and milk fat globule membrane (MFGM) proteins. To mimic human milk's lipid profile, MFGs/MFGM ingredients from sweet whey or buttermilk are fortified in IF. Understanding the structural and compositional, and techno-functionalities changes of MFGs/MFGM is crucial for their application. The MFGs/MFGM has been reconstructed by the mostly used processing methods in dairy industry, acidification (pH 6.30 and 5.30, 25℃), and novel non-thermal treatment, ultrasonic treatments with different intensities. Additionally, anionic polysaccharide- high-methoxyl pectin (HMP) was incorporated to the raw milk before the cream separation, examining the resulting physicochemical properties and techno-functionalities. Results indicated that pH adjustment affected the MFGs structure and MFGM protein compositions, particularly at pH 5.30, causing protein loss such as butyrophilin (BTN) and Periodic Acid Schiff 6/7 (PAS 6/7) and particle size changes. Fourier Transform Infrared Spectroscopy (FTIR) spectra revealed more proteins compositions in MFGs/MFGM prepared from sweet whey (MFGM-C) compared to bovine raw milk, showing lower interfacial tension and ζ- potential. In addition, there were no apparent polymorphs in MFGM-C, and lower exothermic or endothermic enthalpy was needed during the heating and quenching procedures. Acidification, especially pH 6.30 conditions, led to the adsorption of non-membrane milk proteins on the MFGs or MFGM fragments and formed more types of conjugate detected by FTIR. Consequently, this induced changes in the crystal form proportion and thermal behaviour of MFGs/MFGM at pH 6.30. Different acoustic powers generated by the 20 kHz and 40 kHz independently or synchronously working conditions, affected the interfacial properties and thermal behaviour of MFGs/MFGM samples. 20 kHz treatments led to a relatively even particle distribution in MFGs/MFGM compared to 40 kHz and 20 + 40 kHz treated MFGs/MFGM. Ultrasound treatment significantly resulted in the decrease of interfacial tension (π) in all MFGs/MFGM (p0.05). Three typical crystal forms, α-crystal, β-form and β'-form, were observed in control and ultrasonicated 5 min MFGs/MFGM, but α-crystal disappeared in all the 15 min treated MFGs/MFGM. Ultrasonic duration has a more profound effect on the increase of β'- crystal than the intensity. Ultrasonic treatments promoted the crystallisation of MFGs/MFGM fat at higher temperature but increased the melting temperature of MFGs/MFGM. Additionally, the 15 min treatments caused more pronounced changes in the intramolecular β-sheet, random coil, and α-helix structures compared to the 5 min treatments. 20 + 40 kHz/ 15 min treatment led to aggregated membrane proteins indicated by the confocal laser scanning microscopy (CLSM) images. All the MFGs/MFGM exhibited shear-thinning behaviour, except for the 40 kHz treated MFGs/MFGM, which displayed non-Newtonian behaviour at the lower shear rate. The preliminary investigation on the effects of HMP incorporation on the MFGs/MFGM materials showed the enhanced absorbance at the Amide I and Amide II bands and altered fingerprint regions among all the MFGs/MFGM. Larger particle sizes ranging from 10 to 100 μm presented in the 0.30% HMP MFGs/MFGM, suggesting the formation of conjugates between HMP and milk proteins. These structural and compositional changes induced by HMP affected the physical stability and rheological properties of MFGs/MFGM. Notably, MFGs/MFGM with 0.30% HMP exhibited the poorest stability, a significant increase in apparent viscosity, and shear-thinning behavior throughout the analysis. In summary, acidification, ultrasonic treatments, and HMP incorporation significantly impact the structural, compositional, and functional properties of MFGs/MFGM ingredients.
