Food Institute - Doctoral Theses

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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.
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    FODMAPs in cereals, pseudo cereals and legumes: a systematic approach for the development of functional low FODMAP products
    (University College Cork, 2021-07-28) Ispiryan, Lilit; Arendt, Elke K.; Department of Agriculture, Food and the Marine, Ireland
    FODMAPs (fermentable mono-, di, oligosaccharides and polyols) are dietary carbohydrates that have been identified as triggers of symptoms of irritable bowel syndrome (IBS); a diet with a reduced intake of FODMAPs is successfully alleviating symptoms in > 70 % of IBS patients. While whole grain cereals, pulses and products made from those are an essential part of a healthy plant-centered diet, they are also a major source of FODMAPs. Hence, with a lack of functional products with lowered FODMAP contents on the global market, the development of such is emerging in food science and industry. Firstly, an accurate and efficient analytical method for the quantification of FODMAPs, using one single analytical approach (high-performance anion-exchange chromatography coupled with pulsed amperometric detection; HPAEC-PAD), was developed, and served as analytical tool throughout further studies. The FODMAP-profiles of a broad range of cereal-product ingredients, including different cereals, pseudo cereals, gluten-free flours, pulses, pulse protein ingredients, commercial sprouts, and isolates were characterised. Two main classes of FODMAPs were found in the ingredients: fructans in gluten-containing cereals (wheat, spelt, barley, rye) and α-galactooligosaccharides (GOS) in pulses (peas, lentils, chickpeas, etc.). Isolates and fractions from different raw material (pulse protein ingredients, different starches, gluten) had varying GOS or fructan contents, depending on their production process. Gluten-free product ingredients (e.g., rice, millet, buckwheat) did not contain any of the FODMAPs commonly investigated. However, buckwheat accumulates other soluble indigestible carbohydrates (fagopyritols) that may act as FODMAPs. Six ingredients were selected to investigate the impact of malting on FODMAPs: wheat and barley (high in fructans), chickpeas and lentils (high in GOS), oat and buckwheat (‘low in FODMAPs’). In the pulses GOS levels diminished by 80 − 90 % upon the malting process; also, buckwheat fagopyritols were degraded. In contrast, fructan contents in barley and wheat malts were slightly elevated; 0.8 % fructans were even de novo synthesised in oat malt. Finally, aiming for the degradation of fructans and the production of a low FODMAP whole wheat bread, the application of yeast fermentation was investigated. The screening of the FODMAP degradation capability of various yeast isolates led to the selection of two promising strains: Lachancea fermentati FST 5.1 and Cyberlindnera fabianii NTCyb. While the latter revealed to be unsuitable for baking application (very low fermentation rate in wheat dough matrix), L. fermentati FST 5.1 outperformed conventional baker’s yeast (Saccharomyces cerevisiae), with a much more efficient fructan degradation and metabolism of the resulting excess fructose. Apart from low FODMAP contents, the resulting bread had optimal quality characteristics (technological as well as sensory attributes) comparable to the baker’s yeast fermented bread. Overall, the fundamental and systematic work of this thesis provides comprehensive and applicable knowledge essential for developing of low FODMAP products.
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    Lactoferrin in infant nutrition: thermal processing, heteroprotein interactions with osteopontin, simulated gastrointestinal digestion and bioactivity
    (University College Cork, 2021-08) Goulding, David A.; O'Mahony, Seamus Anthony; O'Brien, Nora M.; O'Regan, Jonathan; Nestec