Food Institute - Doctoral Theses

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    Evidence-based design, physiological efficacy testing and consumer analysis of an exemplar model of a fruit derived bioactive functional food for use in physical performance and exercise settings
    (University College Cork, 2023) Carey, Conor C.; Lucey, Alice; Doyle, Lorna; Department of Agriculture, Food and the Marine, Ireland
    The overall aim of this thesis is to examine the new product development practices used within the scientific design, development and testing of new nutritional products in the performance nutrition sector. Despite recent growth in the area there is a general lack of publicly available information to guide the incorporation of nutritional science into the processes underpinning the design and development of new performance nutritional products. Performance nutrition provides a promising area for the creation of value-add products using Irish agrifood outputs, a key priority in Irelands Food Vision 2030 strategy (Gov.ie - Food Vision 2030 – A World Leader in Sustainable Food Systems, 2021). In chapter 1, frameworks that were created as part of this PhD thesis to inform and guide the incorporation of nutritional science in the development of new products are introduced. These frameworks are applied throughout the subsequent chapters in the design and development of an exemplar model of a functional food to support post exercise recovery and endurance exercise performance. This exemplar model took the form of a blackberry polyphenol enriched high protein milk. Chapter 2 reviews the literature on new product development and discusses the role of polyphenols and protein in post-exercise recovery and supporting endurance exercise performance. The review confirms the efficacy of dairy protein, particularly in the form of bovine milk, for muscle repair and regeneration post-exercise. RCTs have consistently shown the benefits of consuming between 1.2-1.6g/kg/day of high-quality protein for those undergoing frequent exercise. While there's growing evidence to support the use of plant-derived polyphenols for exercise-induced muscle damage (EIMD) recovery and endurance exercise performance, gaps remain in understanding their mechanisms, dosing, and optimal dietary sources. Chapter 3 presents an online survey that assesses the nutrition priorities, practices and preferences of athletes and active individuals. This survey was designed as part of this thesis following a series of focus group sessions with athletes of various levels and backgrounds. This survey provides key end-user insights into the nutritional wants and needs of athletes and active individuals. Key findings highlight muscle recovery as the primary nutritional priority among the athletic community and reveal a clear preference for food product forms over supplements. The data collected provides invaluable guidance for new product development in sports nutrition and allows the meaningful incorporation of end-user insights into the process. Chapter 4 bridges a crucial knowledge gap by conducting a systematic review and meta-analysis investigating the efficacy of flavonoid-rich polyphenols to support post-exercise recovery. The meta-analysis of 26 randomised controlled trials (RCTs) observed significant benefits in muscle strength and soreness recovery post intensive exercise. However, a notable limitation in the included studies was the inadequate characterisation of the polyphenol subclasses and dosages used, emphasising the need for more detailed reporting in future human studies. Chapter 5 presents an RCT assessing the efficacy of the RubusElite beverage prototype to support post-exercise recovery. Results suggest that this beverage augments recovery of muscle strength and reduces muscle soreness in comparison to both high protein and low protein milks. In collaboration with RubusElite project partners, this RCT includes detailed characterisation of the beverage's polyphenol content and a verification of polyphenol stability within the treatment beverage for the study duration. Chapter 6 further investigates the efficacy of the RubusElite prototype in relation to physical performance during a 15km cycling trial. In contrast to previous findings, no significant improvement in performance or exertion-related measures was observed. However, thorough polyphenol characterisation alongside these findings offers critical insights into the lack of observed benefits and provides direction for the reformulation of the prototype. This also provides considerable insights into future RCT design to reassess the efficacy of the reformulated prototype. In summary, this thesis offers a comprehensive investigation into the development of performance nutrition products through the creation of an exemplar model using novel frameworks which allow for the incorporation of scientific best practice. It provides innovative insights into the role of polyphenols and protein in supporting exercise and is one of the first undertakings to assess their efficacy in combination. By combining rigorous scientific evaluation, consumer insights, and product prototyping, the research offers a comprehensive roadmap for future product development in the rapidly evolving domain of performance nutrition.
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    Investigations of the complex relationships between minerals, pH and heat stability in milk protein systems
    (University College Cork, 2023) Aydogdu, Tugce; O'Mahony, Seamus Anthony; McCarthy, Noel; Teagasc
    The complex composition of milk and derivatives therefrom, mean that its stability in respect to thermal treatment, while well studied, is ever evolving. Non-fat milk powders are rich in protein, of which there may be greater than 4500 different nitrogenous compounds, often present in conjunction with milk minerals. Variations in the total concentration and individual components of proteins and minerals makes dairy science a multifaceted system from which nutritional dairy products emerge, manipulated and produced by processing factors such as temperature, pressure, dehydration, shear and filtration. At the centre of dairy processing lies the most basic element fundamental to solvent chemistry; hydrogen, used as a measure of chemical reactions, but more often taken as a control parameter for physical and sensory properties. Therefore, the objective of this thesis was to advance considerably our knowledge on milk protein and milk mineral systems with regard to three main properties; heat stability, viscosity and pH changes, which have cumulative effects on dairy processing performance and finished product quality and functionality. A novel method of measuring pH at ultra-high temperatures was adapted from the pharmaceutical industry and used to examine the hydrogen ion concentration in dairy systems. For the first time, a non-linear decrease in skim milk pH was shown with increasing temperature from 25 to 140°C. The pH of skim milk decreased from 6.7 at 25°C to 6.1 at 140°C, with this reduction being reversible on cooling. This was not the case for milk permeate, where the pH remained low after sequential heating and cooling, due to irreversible calcium phosphate formation and precipitation. This highlights the stabilizing ability of micellar casein in skim milk against significant levels of calcium phosphate precipitation. However, while precipitation might be reduced, the addition of milk permeate to milk protein concentrate (MPC) resulted in substantial levels of age thickening after evaporation (45% dry matter), whereas MPC with added lactose showed no age related viscosity increase. The former system also had lower pH compared to the latter. Given the significant influence milk permeate had on viscosity of protein solutions, the heat coagulation time of commercial bulk skim milk obtained across the spring period was assessed to determine if the changes in milk composition from early lactation affected heat stability. Type B HCT-pH profiles were shown for bulk milk samples taken in February and March, compared to a type A profile for April milk, with this type A profile continuing for the remainder of the year. This is the first time that a change in HCT profile has been shown for commercial bulk milk samples; although there was no obvious difference in milk composition between any of the samples. Ultrafiltration of the skim milks was performed to obtain milk permeate fractions and was used to swap the retentate obtained in February with the permeate from April milk. This showed that a type B HCT-pH profile in February could be changed to a type A profile by simply swapping the serum phase. The outcomes of the research in this thesis are highly applicable to the thermal processing of complex nutritional formulations (e.g., infant formulas), where mineral additions and substitutions are common practice. The ability to measure pH during UHT processing is certainly a highlight of the work presented herein. In addition, a key new finding is that macro composition, specifically protein and mineral profile, is not always a good predictor of HCT, but that the serum phase of milk has the more substantial effect on heat stability than the colloidal phase on its own. The work presented in the thesis provides novel information to both the dairy industry and academia, in terms of process control through in-line pH measurement, and the fundamental effects of milk serum on protein heat stability.
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    Sensorial, cultural and volatile properties of dairy powders, yoghurt and butter from pasture and non-pasture cow diets
    (University College Cork, 2022-12-14) Zeng, Cheng; O'Sullivan, Maurice; Kerry, Joseph; Kilcawley, Kieran N.; Teagasc
    Significant advances have occurred in the ability to extract and identify volatile aromatic dairy compounds that may influence sensory perception. In this Thesis volatile extraction techniques were optimised and evaluated in order to obtain a more representative volatile profile of selected dairy products, such as; whole milk powder, skim milk powder, yoghurt and salted butter. This information was also utilised with gas chromatography olfactometry (GC-O) and sensory analysis to determine which specific compounds are most likely influencing sensory perception. In addition the impact of cow diet was assessed in terms of the volatile and sensory profile on skim milk powder, whole milk powder and salted butter using milk from cows outdoors on pasture-fed diets (such as ryegrass, ryegrass and white clover) and cows indoors on trial mixed rations. Cross cultural sensory analysis was also undertaken on skim milk powder from these diets in order to determine if consumers and trained panellists perceived skim milk powder differently based on diet but also on product familiarity Chapter 1 provides an updated review of traditional and novel sensory methods used to evaluate milk, milk powders, yoghurt, and butter, as well as gas chromatography mass spectrometry and gas chromatography olfactometry extraction techniques to provide more complete profile of volatiles that impact sensory perception. Chapter 2 investigates the impact of pasture and non-pasture cow diets on the volatile cross cultural sensory perception of skim milk powder. The volatile profile and sensory properties of the skim milk powder were influenced by cow diet and Irish, Chinese and USA consumers as well as trained sensory panellists perceived products differently primarily based on familiarity. Chapter 3 investigated if yoghurt produced from three different starter cultures were perceived differently by Irish consumers, German consumers and trained assessors. German trained assessors found it more difficult to discern differences between some of the yoghurts than trained Irish assessors. Seventeen of the 24 volatiles compounds identified differed due to starter culture, which most directly or indirectly associated with lipid oxidation.The ability of headspace solid phase microextraction, thermal desorption, and high capacity sorptive extraction as a direct immersion and headspace extraction technique were compared with and without salting out and by a polar and non-polar gas chromatograph column for volatile profiling of whole milk powder in Chapter 4. The impact of three different diets on the sensory properties and volatile profile of whole milk powder was investigated in Chapter 5. Both the sensory perception and volatile profiles of whole milk powder differed significantly depending on the diet, with whole milk powder derived from rye-grass or rye-grass and white clover more similar than whole milk powder derived from total mixed ration. Most of the differences in volatiles due to diet were either directly or indirectly linked to fatty acid content. Chapter 6 outlines the development and optimisation of direct immersion high capacity sorptive extraction for the extraction, separation and identification of volatile compounds from salted butter from three different diets; rye-grass or rye-grass and white clover or from total mixed ration. This thesis has clearly demonstrated that cow diet influences the volatile and sensory characteristics of selected dairy products, which subsequently effects sensory perception on a cultural basis influenced by product familiarity. The benefits of optimising volatile extraction techniques on a product specific basis were clearly demonstrated along with using multiple techniques in order to achieve the most representative volatile profile as possible. Combining volatile analysis with olfactometry and / or sensory techniques enables a more comprehensive understanding of factors influencing sensory perception and choice that can be utilised for product quality, improvement and marketing.
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    Evaluation of the manufacture of cheese from micellar casein concentrate or using novel coagulants
    (University College Cork, 2022-09-23) Li, Bozhao; McSweeney, Paul L. H.; Kelly, Alan; Department of Agriculture, Food and the Marine, Ireland; Food Institutional Research Measure
    Novel materials and coagulants for cheese manufacture are currently of interest since the development of membrane filtration technology and gene recombination technology may offer opportunities for innovation in cheese manufacture. A novel dairy material – micellar casein concentrate (MCC) – is the co-product of whey protein recovery. As the main protein source in cheese is casein, MCC has the potential to be a starting material for cheese manufacture. The objective of the work presented in the first part of this thesis was to evaluate the feasibility of the manufacture of Cheddar and Quarg cheeses from micellar casein concentrate. In addition, camel chymosin has been reported to cause less proteolysis as a coagulant for cheese manufacture compared to bovine chymosin. The suitability of manufacture of Cheddar cheese using a novel camel chymosin with structural changes was also investigated. The rennet and acid coagulation properties of micellar casein concentrate were evaluated. MCC had a higher casein in total protein content compared to low heat skim milk powder (LHSMP), and shorter rennet coagulation time and higher gel strength were found in MCC compared to that of LHSMP. A gelation pH value greater than 5 was found in MCC. MCC produced by cold microfiltration (MF) formed acid-induced gels with high strength at pH 4.6, while the gel strength of acid-induced gels formed by warm MF MCC reached the highest at a pH value of around 5 and decreased below this value due to rearrangements of the casein network. The suitability of the manufacture of Cheddar cheese from MCC was subsequently investigated; standard control milk, skim milk with cream, reconstituted MCC with cream and reconstituted LHSMP with cream were used for comparison. The use of MCC led to increased proteolysis compared to the other treatments, linked to higher plasmin and chymosin activities in the cheese. Increased springiness, cohesiveness and meltability were found in Cheddar cheese manufactured from MCC. For the manufacture of Quarg cheese, lower moisture and higher protein contents were found in cheese made from MCC compared to that made from LHSMP. Cheese made from hot MF MCC showed the highest hardness compared to that made from LHSMP or cold MF MCC. Higher glycomacropeptide (GMP) content was found in cheese whey made from MCC. The suitability of manufacture of Cheddar cheese using a modified fermentation-produced camel chymosin (mCC) was investigated; fermentation-produced bovine chymosin (BC) and camel chymosin (CC) were used for comparison. The use of mCC led to reduced proteolysis compared with BC or CC, and higher instrumental and sensory hardness and lower meltability were found in cheeses made using CC or mCC compared to BC. Descriptive sensory analysis indicated less sulphur and barny flavour in cheese made with CC and mCC, while cheese made using mCC showed the lowest brothy flavour and bitter taste. Finally, the proteolytic specificity of the three generations of chymosin on NaCN at pH 5.2 with 5% NaCl and 6.5 and in proteolysis of Cheddar cheese made using these coagulants were investigated. Many peptides were identified through liquid chromatography-mass spectrometry (LC-MS) in both NaCN digests and Cheddar cheese made using each chymosin. Other than the majority of peptides produced by BC and CC reported in the literature, some new peptides were identified in this study as well. The proteolytic activity of mCC was relatively lower than that of BC and CC. Overall, the results presented in this thesis will support the innovation and application of new materials for the manufacture of cheese and other dairy products and add to the understanding of the properties of three generations of chymosin when used in cheese manufacture.
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    Application of direct and indirect strain engineering approaches to unlock the potential of the yeasts Zygosaccharomyces parabailii and Kluyveromyces marxianus for bio-based processes
    (University College Cork, 2022-05-31) Jayaprakash, Pooja; Morrissey, John P.; Branduardi, Paola; Horizon 2020
    The efficient implementation of biorefineries to produce bio-based chemicals and fuels requires a sustainable source of feedstock and robust microbial factories. Among others, lignocellulosic biomass represents cheap and sugar-enriched feedstock. The conversion of lignocellulosic biomass into the desired products using microbial cell factories is a promising option to replace the fossil based petrochemical refinery. Minimum nutritional requirements and robustness have made yeasts a class of microbial hosts widely employed in industrial biotechnology, exploiting their natural abilities as well as genetically acquired pathways for the production of natural and recombinant products, including bulk chemicals such as organic acids. In bio-based industrial processes, microorganisms are subjected to different kinds of stresses associated with process conditions. These stressors are known to inhibit cellular metabolism and compromise the performances of a fermentative process, being an important limitation to an effective marketability of biobased microbial products. Therefore, the exploration of yeast biodiversity to exploit unique native features and the understanding of mechanisms to endure harsh conditions are essential to develop viable and competitive bioprocesses. In the chapter 1 of this thesis we reviewed the link between LCBs composition, choice of enzymatic cocktail and selection of yeast species and strains that need to be considered in an integrated fashion to enable the development of an efficient bio-based process. We discussed the pivotal role of enzymatic cocktail optimization to unlock the potential of non-Conventional yeasts, which, thanks to broader substrate utilization, inhibitor resistance and peculiar metabolism, can widen the array of feedstock and products of biorefineries. The aim of this PhD work was to expand the industrial potential of two non-conventional yeasts, Zygosaccharomyces parabailii and Kluyveromyces marxianus, by applying direct and indirect strain engineering approaches. These yeasts possess desirable characteristics. K. marxianus has broad specificity for both hexose and pentose sugars as carbon and energy source. Apart from this, its thermotolerance, fast growth and the ability to thrive at pH below 3, make it ideal for industrial use. However, the lack of tolerance of this yeast to inhibitory compounds, particularly weak organic acid produced during LCB pretreatment, hinders its use when this biomass is used as substrates. Although the use of synthetic biology techniques has started to be employed to understand the robustness of K. marxianus and for the production of various chemicals, the mechanisms related to organic acid tolerance are yet to be deciphered. To match this goal, we used Adaptive Laboratory Evolution (ALE), an indirect strain engineering approach, alternative and often complementary to direct engineering. In chapter 2, we aimed to improve the tolerance of K. marxianus to sugar beet pulp (SBP) hydrolysate at pH 3.0 at two different temperatures, 30 oC and 40 oC. Using the ALE approach, we selected K. marxianus evolved isolates with robust phenotype compared to the parental strains, at 30 oC. Differently to K. marxianus, the hybrid yeast Z. parabailii exhibits resistance to weak organic acids (WOA) also at low pH. Understanding the mechanism involved in tolerance to WOA can be used for avoiding the growth of this yeast in food production pipelines as well as for promoting its use as a cell factory for the production of organic acids and other bio-products. In chapter 3 of this study, our aim was to understand the phenotype-genotype correlation involved in the WOA tolerance trait. Using direct engineering method, we constructed and characterised single and double Z. parabailii pdr12 mutants. This study revealed that Pdr12p is involved in tolerance to acetic and butyric acids and not in tolerance towards sorbic and benzoic acids. Furthermore, analysis of the Pdr12p sequence provided insights in the amino acids differences. The pdr12 mutants were constructed by the classical tool of exploiting deletion cassettes. Advances in metabolic engineering and synthetic biology have increased the need for creating techniques such as CRISPR-Cas9 for faster and more efficient genome editing. In chapter 4 of this study our aim was to develop a CRISPR-Cas9 system for simultaneous disruption or deletion of two alleles of a gene in Z. parabailii. We evaluated the use of four different gRNA expression systems consisting of combinations of tRNAs, tRNA and ribozyme or ribozymes as self-cleaving flanking elements. The functionality of the gRNA systems was tested by analyzing the inactivation of the ADE2 gene in the wild type strain and the most efficient gRNA system was used to successfully construct a Z. parabailii dnl4 mutant. This mutant exhibited improved homologous recombination in the deletion of both ADE2 alleles. Analysis of mutations in the gRNA target regions of both ADE2 and DNL4 genes suggested inter-allelic rearrangements between the two gene loci, as well as absence of large regions of chromosomes. Overall, this work contributes to the vast array of studies that are shedding light on yeasts biodiversity, both as a way for understanding their natural potential and as an instrument for tailoring novel cell factories.