Food Institute - Doctoral Theses

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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.
Discovery and molecular characterisation of novel bacteriocins produced by Gram positive bacteria
(University College Cork, 2021-01-08) O'Connor, Paula M.; Ross, R. Paul; Hill, Colin; Science Foundation Ireland
Bacteriocins are ribosomally synthesised antimicrobial peptides, produced by many bacterial genera that display potent activity against closely (narrow spectrum) or distantly related (broad-spectrum) bacteria. Bacteriocins, produced by Lactic Acid Bacteria (LAB) that are natural constituents of fermented foods, are ideal natural preservatives to control food spoilage/pathogenic bacteria in minimally processed foods. In addition to their role as food preservatives, bacteriocins have potent activity against medically significant pathogens and are considered attractive alternatives or adjuncts to antibiotics, due to their inherent heat stability, potency at nanomolar scale, resistance to proteases and low levels of acquired resistance in commercial applications. Overall, bacteriocins are versatile antimicrobials with huge potential for use as biopreservatives, antibiotic alternatives, health promoting gut modulators and animal growth promotors. The aim of this thesis was to identify, purify and characterise novel bacteriocins from microorganisms isolated from a wide range of niches, with a view to expanding the number of bacteriocins currently available and exploring novel structures and activities. In this respect, Chapter 2.1 describes the discovery a novel nisin A variant, nisin H, produced by a porcine gut isolate Streptococcus hyointestinalis DPC6484. Nisin H differs from nisin A at five amino acid positions and is an intermediate between naturally occurring nisins of lactococcal and streptococcal origin. The operon encoding nisin H is noteworthy by virtue of the absence of an equivalent of nisI that encodes an immunity protein that protects the cell from its own bacteriocin. This is the first report of natural nisin variant production by an intestinal isolate of streptococcal origin and may confer an advantage to the strain by allowing it to dominate its environment, fight infection or signal the immune system of the host. In a subsequent chapter another natural variant is characterised in the form of nisin J, produced by a human skin isolate Staphylococcus capitis APC2923. Nisin J is more dissimilar to nisin A than nisin H with nine amino acid changes, six of which are unique, and an extra amino acid making it the first nisin variant to contain 35 amino acids. Interestingly, the operon lacks both nisI (immunity) and nisRK (regulatory) equivalents. Nisin J, like nisin A and H, displays activity against a wide number of genera and represents the first natural nisin variant from staphylococci and the first nisin producer from human skin, suggesting a role in competitive colonization for producing organisms. The natural nisin variants described above (nisin H and J), in addition to nisin P produced by Streptococcus agalactiae DPC7040, are all produced by non GRAS strains and are therefore limited in their potential industrial applications. The recent increase in the prevalence of antibiotic resistant pathogens makes it important that all bacteriocins regardless of the producing organism are explored as antibiotic alternatives. As these lantibiotics are gene encoded, bioengineering (Chapter 3.1) was used to enable recombinant expression of peptides naturally expressed by non-GRAS organisms in a host derived from safe origins. Specifically, the Nisin A promotor and nisin A leader sequence were fused to nisin H, J or P structural genes and successfully expressed in the GRAS strain L. lactis NZ9700, demonstrating that the L. lactis production, transport and modification machinery can produce fully functional nisin variants from significantly different genetic backgrounds. In Chapter 4, Bactofencin A produced by Streptococcus salivarius DPC6502 was discovered following a porcine gut mining study. It is a 22 amino acid, class IId bacteriocin that displays activity against Staphylococcus aureus and Listeria monocytogenes. Structurally, it consists of a positively charged N terminus that we propose could bind to the negatively charged cell surface. The small bacteriocin cluster also encodes a DltB homologue that may well be responsible for immunity through D-alanylation of teichoic acids. In order to probe structure/function relationships in bactofencin A, a library of synthetic bactofencin A peptide variants were synthesized. Substituting cysteine residues significantly reduced activity confirming the importance of the disulphide while sequential removal of the positively charged N terminal resulted in a decreasingly active peptide. Substituting each amino acid for alanine revealed that residues 9-17 within the loop were more affected by substitution, suggesting this region contributes significantly to the potency of the bacteriocin. In Chapter 5, bactofencin A was shown to enhance nisin bactericidal activity and reduce the overall frequency of resistance. Interestingly, these studies highlighted the relatively slow or delayed mode of action of bactofencin A. The last two chapters (Chapters 6.1 and 6.2) again focus on the discovery of two novel bacteriocins, namely formicin and actifensin. The first of these, formicin, is a novel bacteriocin that extends the class of two peptide lantibiotics. It was purified from Bacillus paralicheniformis APC1576, a mackerel intestine isolate. Compared with other two component lantibiotics, formicin is most similar to haloduracin and consists of a very hydrophilic Alpha peptide with a charge of +2 whereas the Beta peptide is negatively charged. Formicin displays activity against a broad range of Gram-positive bacteria including clinically relevant pathogens. The second bacteriocin is actifensin a 4091 Da, broad spectrum, Class IId bacteriocin containing three disulphide bridges with more than 50% similarity to eukaryotic defensins that we propose represents a new subclass of bacteriocins. It is produced by Actinomyces ruminicola, isolated from sheep feces. A pangenomic screen of available Actinomyces spp. revealed the presence of very diverse actifensin homologues in 29% of genomes examined, suggesting that production of actifensin like bacteriocins is a common trait. This new class of bacteriocins may provide a template to design new broad-spectrum antimicrobials for treatment of human and animal infections. The developments described in this thesis can be used to contribute to increased commercialisation of bacteriocins in both food systems and human and animal medical treatments.
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.
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.
Fundamental investigation and applied studies on non-Saccharomyces yeasts in non-alcoholic and low alcohol beer brewing
(University College Cork, 2019-12-11) Bellut, Konstantin; Arendt, Elke K.; Fonds Baillet Latour
Non-alcoholic and low alcohol beer (NABLAB) is enjoying growing popularity owing to consumer lifestyle changes, improved production methods and stricter legislation. Among the biological methods for their production, particularly research into non-Saccharomyces yeasts has gained momentum in recent years in order to produce NABLAB with novel flavor characteristics in an easy-to-apply manner. In a proof-of-concept study, five selected non-Saccharomyces species isolated from kombucha showed to perform just as well in laboratory-scale trials in wort as commercially applied species Saccharomycodes ludwigii. In a subsequent study, species of the Cyberlindnera genus were found to produce a pleasant, fruity flavor in wort. Fermentation parameters were optimized by means of response surface methodology (RSM) and the resulting non-alcoholic beer (NAB; 0.36% ABV) produced with Cyberlindnera subsufficiens on pilot-scale (60 L) had a significantly more fruity and significantly less wort-like aroma compared to two commercial NABs. Regarding low alcohol beer (LAB), the yeast species Lachancea fermentati was introduced to create LAB by harnessing the species’ uncommon ability to produce significant amounts of lactic acid (LA) during alcoholic fermentation. Compared to a Saccharomyces cerevisiae brewers’ yeast, L. fermentati produced less ethanol (–15%) while producing 1.3 g/L lactic acid, giving the beer a sour taste. In a follow-up study, four L. fermentati isolated from individual kombucha cultures were investigated in detail. The strains genotypes and phenotypes where shown to be diverse, correlating with the strains’ geographical origin. LA production was optimized via RSM, where low pitching rate, high fermentation temperature, and a high initial glucose concentration resulted in the highest LA concentrations (max. 1.6 g/L). LAB (1.26 %ABV) produced with L. fermentati by stopped fermentation showed to have a balanced ratio of acidity from lactic acid to residual wort sweetness. In conclusion, the results of this thesis give prospect to future studies with non-Saccharomyces yeasts and strengthen their position as a serious and applicable alternative to established methods in NABLAB brewing.
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
Omics based approaches for the identification of novel bioactive secondary metabolites from marine sponge derived bacterial isolates
(University College Cork, 2021-02) Patry, Sloane; Dobson, Alan; Horizon 2020
Antibiotic resistance is a major threat to public health worldwide which, urgently requires the discovery and development of novel antimicrobial drugs with new modes of action. The principal goal of this project is the discovery of new bioactive secondary metabolites that may lead to the discovery of new antibiotics to help fight against the antibiotic resistance crisis. The marine environment is a unique habitat where deep-sea microorganisms, because of their ability to adapt to this extreme environment, have the potential to produce novel secondary metabolites with potent biological activities. Furthermore, microbes associated with marine sponges are exposed to highly competitive environments both physiologically and nutritionally which is likely to promote the production of novel secondary metabolites. The genus Streptomyces produces secondary metabolic compounds that are rich in biological activity and marine Streptomyces strains host abundant small molecule biosynthetic gene clusters (smBGCs) which encode polyketides, Non ribosomal peptide synthases (NRPS), siderophore, bacteriocin and lantipeptides. In this respect, by focusing on Streptomyces samples taken from sponges in marine environments off the Irish coast, we aimed to characterize new marine derived bacterial strains and to explore their bioactive potential. In order to maximize the number of potential novel bioactive metabolites that could be be identified from each Streptomyces strain, and to generate the most detailed information underpinning their associated metabolic pathways, the One Strain MAny Compounds (OSMAC) approach, combined with a multi-omics approach, was employed in this project. In total, the Streptomyces isolates (B226SN104, SM3 and SM9), produced 612 metabolites including but not limited to, Bisucaberin B, Maculosin, Desferrioxamine B, Desferrioxamine E, Concanamycin B, Lipoamicoumacin B, Probestin, Surugamide A, Fiscalin B, Gibbestatin B and Antimycin A1. The Streptomyces isolates produced metabolites active against the growth 21 of antimicrobial-resistant microorganisms categorized as an “Urgent” or “Serious” threat by the World Health Organization (WHO), including Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus and Candida spp. In addition, other activities of biomedical interest were identified, namely anti-cancer, anti-inflammatory and antioxidant activities. Furthermore, the omics approach allowed us to predict 19 potential bioactive and potential novel promising molecules from the Streptomyces SM9 isolate. Taken together, this project demonstrated the significant potential the modified OSMAC/multi omics approach could have in expanding the number of novel secondary and bioactive metabolites that could be generated from existing environmental microbial isolates. Furthermore, it has contributed to the fight against antimicrobial resistance by identifying as of yet potential undiscovered molecules with antimicrobial potential.
Patterns of proteolysis in Danish blue, Camembert, smear-ripened and Cheddar cheeses
(University College Cork, 2020-06-04) Mane, Anuya; McSweeney, Paul L. H.; Food for Health (Ireland)
Patterns of proteolysis in Danish blue, Camembert, smear-ripened and Cheddar cheeses The overall aim of this thesis was to investigate patterns of proteolysis in Cheddar, Danish blue (normal and treated), Camembert and smear ripened cheeses at different stages of ripening. Proteolysis in Danish blue was studied during 9 weeks of ripening, specificities of Penicillium roqueforti proteinases on the caseins in milk hydrolysates were determined with a total of 91 and 118 cleavage sites identified in s1- and -casein, respectively. Proteolysis in an Irish farm-house Camembert cheese was studied during 10 weeks of ripening, specificities of Penicillium camemberti proteinases on the caseins in milk hydrolysates were determined and 64, 6, 28 and 2 cleavage sites were identified in s1-, s2-, - and -casein, respectively. Proteolytic, physico-chemical and lipolytic properties of Danish blue cheeses made from partially (73%) or fully homogenised milk (200 L) with or without whey cream were determined at 4 weeks and 8 weeks of ripening. The study showed that incorporation of the whey cream did not cause any major difference to the compositional, proteolytic or lipolytic properties of Danish blue cheese. Proteolysis in an Irish farmhouse smear-ripened cheese was studied by serial slicing (0.41 mm/slice) the first 2 cm from surface towards the center of the cheese. Peptides produced at depths 0.41 mm and 20.5 mm were 720 and 427 from αs1-casein; 691 and 337 from αs2-casein; 807 and 453 from-casein; 180 and 109 from -casein, respectively. The study confirmed higher proteolytic activity at the surface due to action of enzymes of the smear microbiota than at the center of the cheese. Secondary proteolytic changes of the chymosin-derived peptides s1-CN (f1-23) and -CN (193-209) were investigated in Cheddar cheeses made using single strains of Lactococcus lactis subsp. cremoris HP and SK11 during 6 months of ripening and proteolytic specificities of the cell-envelope proteinases of these strains on the caseins were determined. A large number of peptides were identified from mass spectrometric analysis of the soluble extracts of the individual cheesees and highest values of relative intensity from the mass spectrometric analyses were used to plot the cleavage sites that can be used for further understanding of peptides, cleavage site and similar research.
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.
Studies on selected physicochemical properties and microstructure of β-casein-enriched ingredients for applications in formulated nutritional products
(University College Cork, 2020-09) Li, Meng; O'Mahony, Seamus Anthony; Kelly, Alan; Brodkorb, André; Irish Dairy Levy Research Trust; Teagasc Walsh Scholarship Scheme
The milk protein β-casein (β-CN) possesses excellent functional properties and is the most abundant casein in human milk. Therefore, β-CN has the potential to be used as an ingredient in nutritional food applications, in particular in infant formulae (IF). However, the manufacture of β-CN products using various feed materials and enrichment approaches can lead to considerably differences in the overall composition and purity of β-CN in such ingredients. These differences would be expected to influence the physicochemical and functional properties of β-CN ingredients and their formulated nutritional products. This thesis presents an investigation of the physicochemical and microstructural properties of three different β-CN products (pure β-CN [β-CNpure, β-CN=90% of total protein], β-CN concentrate [β-CNconc, 80% purity], β-CN-enriched ingredient [β-CNen, 58% purity]) in different dairy systems (solutions, emulsions and model IF). The results of this work demonstrated that the three β-CN products exhibited different thermal-induced association behaviour, which was highly dependent on the purity and composition of β-CN products, along with the presence of ionic calcium and phosphates. Both β-CNpure and β-CNen showed significantly lower surface/interfacial tension compared to whey protein isolate (WPI), indicating the excellent surface-active properties of β-CN. The stability of 10% oil-in-water (O/W) emulsions stabilised with 0.5% β-CNconc was strongly dependent on the state of association of β-CN in aqueous solution. β-CNpure was covalently labelled using a fluorescent dye (NHS-Rhodamine) and the mixture of labelled β-CN and WPI (1:1 w/w) was employed as an emulsifier in O/W emulsions. Combining covalent and generic labelling techniques with confocal laser scanning microscopy (CLSM) allowed simultaneous visualisation of β-CN and WPI at the interface of oil droplets. β-CN-enriched model IF were also produced with β-CNen and WPI at various whey protein:casein ratios in the range 100:0-0:100, where the model IF enriched with β-CNen better reflected the protein profile of human milk. Increasing the proportion of β-CNen increased the stability and viscosity of IF and decreased the wettability and dispersibility of β-CNen IF powders. Overall, the findings of these studies are of relevance to end-users of β-CN-enriched products in controlling the association behaviour of β-CN and improving the stability of nutritional products enriched with β-CN.
Translating plant protein benefits to consumer products: physicochemical, technological and nutritional aspects of utilising plant protein ingredients for cereal food applications
(University College Cork, 2021-03) Hoehnel, Andrea; Arendt, Elke K.; Stanton, Catherine; Horizon 2020 Framework Programme
The way we currently produce and consume food puts both environmental sustainability and human health at great risk. A global shift to predominantly plant-based diets as part of a food system transformation promises remedies for environmental implications as well as rising diet-related disease risks. Nonetheless, a decreased consumption of animal-based foods and increased inclusion of plant-based protein in our diets entails challenges with regard to the development of plant-based protein-foods and their convenience, technological quality and nutritional quality, which were addressed in this thesis. A thorough physicochemical characterisation of plant-based high-protein ingredients (HPIs) is important to explore their full potential for human nutrition and to identify appropriate processing techniques. For example, the quantification of short-chain carbohydrates in plant-based HPIs is required when fermentation technology is applied. In a study comparing commonly used aqueous extraction methods for short-chain carbohydrates with alternative ethanolic extraction methods, ethanolic extraction was found to be superior. Due to high water absorption capacity and gelation, aqueous extraction is often not applicable to plant-based HPIs from a practical perspective. Furthermore, enzymatic activity causing conversion or degradation of short-chain carbohydrates can be minimised with ethanolic extraction. Cereal-based staple foods, like bread and pasta, represent promising matrices for the incorporation of plant-based HPIs (e.g. partial replacement of wheat flour or wheat semolina) and offer the opportunity to provide plant-based protein-foods in convenient formats with high consumer acceptance. In a fundamental bread application study, several plant-based HPIs (derived from cereals, potato, legumes) were screened for their baking performance in high-protein breads (> 20% of calories provided by protein). Based on the findings of this study, an optimised high-protein bread formulation (containing faba bean and carob HPIs) with technological quality and sensory attributes similar to regular wheat bread and improved nutritional profile was proposed. This high-protein hybrid bread was characterised by an isocaloric replacement of wheat starch by non-cereal protein (starch content - 21%, which indicates reduced glycaemic load) and an improved amino acid balance (lysine content based on protein + 65%). Also, a substantially improved nitrogen utilisation (+ 69%) and protein efficiency ratio (+ 88%) compared to regular wheat bread were found in in vivo nitrogen balance tests. Fermentation (utilising the strain Leuconostoc citreum TR116) was investigated as a tool to further improve HPIs’ baking performance and proven to successfully functionalise faba bean ingredients for bread applications, for example, by improving gluten-aggregation, bread volume (+ 19–22%) and crumb hardness (- 38–49%) in the presence of fermented (instead of unfermented) faba bean ingredients. For pasta applications, the combination of HPIs derived from pseudocereals (buckwheat) and legumes (lupin, faba bean) was particularly beneficial to achieve good technological quality since the HPIs were shown to compensate each others’ (positive and negative) effects on quality characteristics like cooking loss, stickiness and tensile strength. The improvement of the high-protein hybrid pasta’s nutritional quality (compared to regular wheat pasta) was also quantified and found to be similar to that observed for high-protein hybrid bread (compared to regular wheat bread).
Understanding aroma and flavour formation in baked confectionery products, as influenced by sugar and fat
(University College Cork, 2020-12-18) Garvey, Emer; Kerry, Joseph; O'Sullivan, Maurice; Kilcawley, Kieran; Department of Agriculture, Food and the Marine, Ireland; Food Institutional Research Measure
This thesis directly addresses consumer demands for ‘clean-label’, sustainable and natural ingredients in baked confectionery products. Baked confectionery products are a prime matrix to explore alternative sucrose replacers due to the critical functionality of sucrose and fat in relation to desirable structural and organoleptic properties. The primary objective of this research was to elucidate the impact of modifying sugar and butter on the aromatic and sensory properties of selected baked confectionery products. Chapter 1 provides an updated review of reduced fat and sucrose research in baked confectionery products focussing on the association of aromatic volatiles and sensory perception. Chapter 2 outlines the development and optimisation of a headspace solid-phase microextraction gas-chromatography mass-spectrometry (HS-SPME-GC-MS) method for the extraction, separation and identification of volatile compounds from a baked confectionery matrix. Extraction parameters were optimised and the method was validated and applied throughout all subsequent volatile analysis in this thesis. Chapter 3 explored the influence of clean-label sucrose replacers on the sensory quality and volatile aroma of sponge cakes. Gas-chromatography-olfactometry (GC-O) was applied to assess the impact of sucrose replacers on aroma perception, in combination with consumer evaluation and ranking descriptive analysis (RDA) to better understand changes in sensory perception. The influence of sucrose particle size, and sucrose source (beet or cane sugar) in sponge cakes was explored in Chapter 4. Chapter 5 investigated if butter produced from pasture and non-pasture bovine diets, had different sensory characteristics, in a cross-cultural context. Consumer studies were conducted in Ireland, Germany and the USA, and, RDA was conducted in Ireland and Germany, with descriptive analysis (DA) carried out in the USA. The butters produced from these same bovine diets were incorporated into shortbread biscuits, and liking was evaluated using consumers, in additional to temporal assessment, using temporal-check-all-that-applies (TCATA) to further understand the impact on the sensory properties of the shortbread biscuits (Chapter 6). In summary this research demonstrated that the composition of sucrose replacers (particularly when containing reducing sugars), can accelerate Maillard (MR) and Caramelisation (CR) reactions, influencing sensory perception. ‘Spicy/bready’ furfural contributed most to the overall aroma of the sponge cake samples, and that ‘fatty cake crust’ heptanal and ‘potato damp’ methional, varied most between the control (100% sugar) and the 30% w/w reduced sugar sponge cakes with apple pomace powder and oligofructose, respectively. Sucrose source did not significantly affect (P < 0.05) the volatile profile of sponge cakes; however, reduced sugar crystal size positively influenced MR and CR compounds. No significant difference (P < 0.05) was identified in the overall liking, among USA, German and Irish consumers, of the experimental butters-although cross-cultural preferences were clearly evident. Sensory attribute differences based on cow diet were likely influenced by familiarity. The colour of shortbread biscuits formulated with pasture was perceived more favourable by consumers due to the golden colour, as a result of higher β-carotene content. The temporal profile of the shortbread biscuits, evaluated during the stages of oral processing; orthonasal, in-mouth and aftertaste, differentiated mainly due to variations in the fatty acid composition of the butter, which highlights that relatively minor changes in the fatty acid profile of butter can impact on the sensory characteristics of baked confectionery products where it is used as an ingredient This PhD thesis has highlighted the potential of combining sensory techniques, volatile profiling and olfactometry to provide in-depth information to aid in understanding aroma development in baked confectionery products. This approach can evidently be used to improve the sensory quality of baked confectionery products and especially products with modified sucrose and fat contents; however it is also very applicable to improving the quality of any food type and in new product development.

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