This thesis is focused on determination of fatty acids in processed and/or processed cheese analogues using gas chromatography with FID detection. Characterization of fatty acids, various methods for lipid extraction and the possibilities of determination of fatty acids are described in the theoretical part. Simple and rapid method for the determination of fatty acids in natural and/or processed cheese was optimized and validated in the experimental part. The modified Folch method (by mixture of chloroform - methanol) was selected for lipid extraction from the samples. The standard method (ISO 5509), using a methanol solution of potassium hydroxide, was selected for lipid esterification. Type of the stationary phase and parameters of the column, flow of carrier gas, temperature of the injector, injection technique, temperature of the detector and temperature program were optimized. Calibration curves were constructed from five calibration points (in the concentration range of 0.01 - 60 g.ml-1), all correlation coefficients R2 were higher than 0,99. The limits of detection and quantification of individual fatty acids ranged from 0.002 - 6 g.ml-1. The repeatability of the method was good, all the relative standard deviations of the peaks were below 9 %. The optimized and validated method was applied to selected samples of processed cheese analogues. In total five types of analogues containing different kinds of fat (butter, apricot, linseed, black currant and grape seed oils) were analyzed. In samples 34 fatty acids were identified based on a comparison of retention times of standards.

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Doctor of Philosophy / Department of Grain Science and Industry / Praveen V. Vadlani / As the world population continues to grow and the uncertainty of petroleum and food availability transpires, alternative resources will be needed to meet our demands. Single cell oil (SCO) from oleaginous yeast is a renewable noncrop-based resource that can be used for the production of petroleum counterparts. Currently, commercial production is limited, mainly due to high production costs and competition from cheaper alternatives. As a result, improved fermentation techniques, utilization of low-valued feedstocks and efficient downstream processing would be highly valuable. The major objectives of this study were to: 1) optimize fermentation conditions for the development of a novel fed-batch fermentation to enhance oil production using Lipomyces starkeyi, 2) determine the major lipids produced by L. starkeyi, 3) utilize low-valued hemicellulose-rich feedstocks for oil production, and 4) demonstrate the use of 2-methyltetrahydrofuran (2-MeTHF) and cyclopentyl methyl ether (CPME) as greener solvents for oil extraction. Under optimized fermentation conditions, the oil yield increased from 78 to 157 mg oil/g sugar when supplying xylose rather than glucose as the major carbon source. A novel repeated fed-batch fermentation supplying glucose for growth and xylose for lipid accumulation generated the highest oil yield of 171 mg oil/g sugar, oil content of 60% (dry mass basis) and oil productivity of 143 mg oil/L/hr. Oleic acid accounted for 70% of the total fatty acid profile indicating that oil from L. starkeyi is a naturally high source of oleic acid; an added benefit for the biofuel, cosmetic, food, and oleochemical industries. Hemicellulose-rich corn bran and wheat bran were successfully used to produce oil; oil yields of 125 and 71 mg oil/g sugar were reported for whole and de-starched bran hydrolysates, respectively. Compared to traditional methods, biphasic oil extraction systems of 2-MeTHF and CPME had an 80 and 53% extraction efficiency and 64 and 49% selectivity, respectively. The information from this study will be useful for the development of an integrated approach to improve the viability of SCO biochemical platforms for the production of advanced biofuels and renewable chemicals.

Orientador: Marcela Aparecida Guerreiro Machado / Resumo: A microalga Chlorella possui ácidos graxos poli-insaturados, vitaminas e alto conteúdo proteico. Desse modo, estudos recentes têm explorado o uso de microalgas para obtenção de lipídios, principalmente os de maior valor comercial como o ácido ɣ-linolênico. Estre trabalho objetivou propor um método de cultivo para a microalga Chlorella vulgaris e extração do óleo. Os experimentos foram realizados em duplicata de acordo com o arranjo ortogonal de Taguchi, a partir da qual foram organizadas duas etapas: na primeira foram viabilizados cultivos nos quais os fatores de interesse pudessem ser avaliados em dois níveis de operação: Aeração (1,67 e 3,33L/min), NO3 (0,25 e 0,50 g/L), PO4 -3 (0,35 e 0,65 g/L), NaHCO3 (0,25 e 0,50 g/L), intensidade de luz (0,85 e 14,50 K luz) e fotoperíodo (12 e 24 h). Em tal etapa correu também a colheita, floculação, secagem e moagem da biomassa. Na segunda etapa foram realizadas as extrações lipídicas de acordo com e uso de solventes como o clorofórmio e metanol, utilizou-se também o banho de ultrassom para as extrações dos teores lipídicos. Obteve melhor resultado a concentração de biomassa algal, a qual atingiu a concentração de nitrato (0,50 g/L), concentração de bicarbonato de sódio (0,50 g/L), fosfato (0,65 g/L) e fotoperíodo de 12 em 12hs. O melhor resultado para a extração lipídica ocorreu conforme a metodologia de Zorn (2017), com aproximadamente 2,5% mais eficiente e obteve o seguinte resultado para os fatores, no nível alto são: aeração (3,33... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The micro-algae Chlorella has polyunsaturated fatty acids, vitamins and high protein content. Thus, recent studies have explored the use of microalgae to obtain lipids, especially those with higher commercial value such as β-linolenic acid. The objective of this work was to propose a method of cultivation for the microalga Chlorella vulgaris and extraction of the oil. The experiments were carried out in duplicate according to Taguchi's orthogonal arrangement, from which two stages were organized: in the first, cultures were feasible in which the factors of interest could be evaluated at two levels of operation: aeration (1.67 e 3.33L/min), PO4 -3 (0.35 and 0.65 g / L), NaHCO 3 (0.25 and 0.50 g / L), NO 3 (0.25 and 0.50 g / L) , light intensity (0.85 and 14.50 K light) and photoperiod (12 and 24 h). In this stage also the harvest, flocculation, drying and milling of the biomass took place. In the second stage, the lipid extractions were performed according to the use of solvents such as chloroform and methanol, and the ultrasonic bath was used to extract the lipid contents. The best results were the algal biomass concentration, which reached the concentration of nitrate (0.50 g / L), sodium bicarbonate (0.50 g / L), phosphate (0.65 g / L) and photoperiod every 12 hours The best result for the lipid extraction occurred according to the Zorn (2017) methodology, with approximately 2.5% more efficient and obtained the following result for the factors, at the high level are: aerati... (Complete abstract click electronic access below) / Mestre

Microbial cell disruption using pressurized gas is a promising approach to improve the lipid extraction yield directly from the wet biomass by eliminating the energy-intensive drying process, which is an integral part of traditional methods. As the process starts with the solubilization of the gas in lipid-rich microbial cells, it is important to understand the solubility of different potential gases in both lipid (triglyceride) and lipid-rich microbial cell culture to design efficient cell disruption processes. In this study, we determined the solubility of different gases (e.g., CO2, CH4, N2, and Ar) in canola oil (triglyceride) using a pressure drop gas apparatus developed in our laboratory. The solubility of different gases in triglyceride followed the trend CO2 > CH4 > Ar > N2. Since the solubility of CO2 was found to be higher compared to other gases, the solubility of CO2 in lipid rich cell culture, cell culture media, and spent media was also determined. It was found that CO2 is more soluble in triglycerides, but less soluble in lipid-rich cell culture compared to CO2 in water. From both thermodynamic models and Monte Carlo simulations, the correlated solubility was found to be in good agreement with the experimental results. CO2 was found to be the most suitable gas for microbial cell disruption because almost 100% cell death occurred when using CO2 whereas more than 85% cells were found to be active after treatment with CH4, N2, and Ar. The optimization of microbial cell disruption was conducted using the combination of Box-Behnken design of experiment (DOE) technique and response surface methodology. The optimized cell disruption conditions were found to be 3900 kPa, 296.5 K, 360 min, and 325 rpm where almost 100% cell death was predicted from the statistical modeling. Finally, it was found that 86% of the total lipid content can be recovered from the wet biomass after treatment with pressurized CO2 under optimized conditions compared to control where up to 74% of the total lipid content can be recovered resulting in 12% increase in the lipid extraction yield using pressurized CO2.

Crèmes, shampooings, savons, gels douche, ces produits cosmétiques quotidiennement utilisés, ont en commun la présence d'une matière première essentielle à leur formulation, à savoir le tensioactif ou émulsionnant. Ces molécules sont donc fréquemment en contact avec la peau. En effet, de par sa structure amphiphile particulière, le tensioactif aide à la stabilisation des émulsions, permet la formation de mousse et apporte les propriétés détergentes des produits cosmétiques nettoyants, en solubilisant les corps gras présents en surface. Ces molécules peuvent également interagir avec les composants de la peau. Une des premières preuves évidentes de l'interaction des tensioactifs avec la peau est l'observation des signes cliniques suite à l'exposition prolongée ou chronique à des formules riches en tensioactifs. Ces molécules sont maintenant connues pour engendrer des dermatites de contact d'irritation et font l'objet d'un véritable problème de santé publique concernant les maladies professionnelles aux détergents. Cependant, devant la multitude de tensioactifs présents sur le marché, les mécanismes d'action des tensioactifs sur la peau sont encore mal connus, surtout pour les tensioactifs non ioniques, très utilisés dans les produits de soin et souvent considérés comme non toxiques. Le sodium lauryl sulfate, tensioactif anionique reste à ce jour, la molécule modèle couramment étudiée. La toxicité cutanée est le plus souvent reliée à l'interaction du tensioactif avec les protéines, molécules chargées. De ce fait, les tensioactifs ioniques sont considérés comme étant les plus toxiques pour la peau. De plus, la forme monomérique du tensioactif est décrite comme l'entité responsable de la toxicité cutanée, s'insérant plus facilement dans la bicouche lipidique et pouvant ainsi pénétrer plus facilement dans la peau que sous la forme de micelles. La toxicité du tensioactif est donc également reliée à sa concentration micellaire critique. L'objectif de ce projet de recherche était d'approfondir les connaissances sur différentes propriétés physicochimiques de nombreuses classes de tensioactifs, afin de mieux comprendre leurs interactions ainsi que leurs effets sur la peau. Plusieurs niveaux d'études ont été développés. Après une analyse physicochimique des tensioactifs détaillée, des mesures in vitro ont permis d'évaluer l'effet du tensioactif sur la toxicité cutanée. L'étude portant sur la fonction barrière de la peau (propriétés de surface/détergence, organisation de la matrice lipidique et évaluation de l'extraction lipidique) a été menée via des expérimentations ex vivo. Nos études ont montré une toxicité notable de certains tensioactifs non ioniques et a contrario certains tensioactifs ioniques se sont révélés parfaitement bien tolérés. Les paramètres soulignés dans la littérature tels que la CMC et la charge des tensioactifs ont été remis en question. Plusieurs explications ont été mises en avant considérant l'organisation du tensioactif dans l'eau et son comportement vis-a-vis de la fonction barrière cutanée apportant ainsi de nouvelles pistes pour une meilleure compréhension de l'effet du tensioactif sur la peau. De plus La toxicité des tensioactifs a pu être reliée à un des trois niveaux de perturbation de la barrière cutanée: la désorganisation de la matrice lipidique / Creams, shampoos, soaps, shower gels, these cosmetics daily used, have in common the presence of a raw material essential to their formulation, namely the surfactant or emulsifier. These molecules are therefore frequently in contact with the skin. Indeed, because of its particular amphiphilic structure, the surfactant helps the stabilization of emulsions, allows the formation of foam and provides the detergent properties of cleansing cosmetic products, by solubilizing the fatty substances present on the surface. These molecules can also interact with the components of the skin. One of the first obvious evidence of the interaction of surfactants with the skin is the observation of clinical signs following prolonged or chronic exposure to surfactant-rich formulas. These molecules are now known to cause irritant contact dermatitis and are the subject of a real public health problem regarding professional detergent diseases. However, considering the multitude of surfactants on the market, the mechanisms of action of surfactants on the skin are still poorly known, especially for nonionic surfactants, widely used in skincare products and often considered non-toxic. Sodium lauryl sulfate, anionic surfactant remains to this day, the model molecule currently studied. The cutaneous toxicity is most often related to the interaction of the surfactant with the proteins, charged molecules. As a result, ionic surfactants are considered to be the most toxic to the skin. In addition, the monomeric form of the surfactant is described as the entity responsible for cutaneous toxicity, fitting more easily into the lipid bilayer and thus able to penetrate the skin more easily than in the form of micelles. The toxicity of the surfactant is therefore also related to its critical micelle concentration. . The objective of this research project was to expand knowledge on different physicochemical properties of many classes of surfactants, to better understand their interactions and their effects on the skin. Several levels of studies have been developed. After detailed physicochemical analysis of the surfactants, in vitro measurements were used to evaluate the effect of the surfactant on skin toxicity. The study on the skin barrier function (surface properties / detergency, lipid matrix organization and evaluation of lipid extraction) was conducted via ex vivo experiments. Our studies have shown significant toxicity of some nonionic surfactants and conversely some ionic surfactants have been found to be perfectly well tolerated. Parameters highlighted in the literature such as CMC and surfactant charges have been questioned. Several explanations were put forward considering the organization of the surfactant in the water and its behavior on the skin barrier function thus bringing new tracks for a better understanding of the effect of the surfactant on the skin. In addition, the toxicity of the surfactants could be related to one of the three levels of disruption of the cutaneous barrier: the disorganization of the lipid matrix

Les peptides et protéines extracteurs de lipides (PEL) se lient aux membranes lipidiques puis en extraient des lipides en formant de plus petits auto-assemblages, un phénomène qui peut aller jusqu'à la fragmentation des membranes. Dans la nature, cette extraction se produit sur une gamme de cellules et entraîne des conséquences variées, comme la modification de la composition de la membrane et la mort de la cellule. Cette thèse se penche sur l’extraction lipidique, ou fragmentation, induite par le peptide mélittine et la protéine Binder-of-SPerm 1 (BSP1) sur des membranes lipidiques modèles. Pour ce faire, des liposomes de différentes compositions sont préparés et incubés avec la mélittine ou la BSP1. L'association aux membranes est déterminée par la fluorescence intrinsèque des PEL, tandis que l'extraction est caractérisée par une plateforme analytique combinant des tests colorimétriques et des analyses en chromatographie en phase liquide et spectrométrie de masse (LCMS). La mélittine fait partie des peptides antimicrobiens cationiques, un groupe de PEL très répandu chez les organismes vivants. Ces peptides sont intéressants du point du vue médical étant donné leur mode d’action qui vise directement les lipides des membranes. Plusieurs de ceux-ci agissent sur les membranes des bactéries selon le mécanisme dit « en tapis », par lequel ils s’adsorbent à leur surface, forment des pores et ultimement causent leur fragmentation. Dans cette thèse, la mélittine est utilisée comme peptide modèle afin d’étudier le mécanisme par lequel les peptides antimicrobiens cationiques fragmentent les membranes. Les résultats montrent que la fragmentation des membranes de phosphatidylcholines (PC) est réduite par une déméthylation graduelle de leur groupement ammonium. L'analyse du matériel fragmenté révèle que les PC sont préférentiellement extraites des membranes, dû à un enrichissement local en PC autour de la mélittine à l'intérieur de la membrane. De plus, un analogue de la mélittine, dont la majorité des résidus cationiques sont neutralisés, est utilisé pour évaluer le rôle du caractère cationique de la mélittine native. La neutralisation augmente l'affinité du peptide pour les membranes neutres et anioniques, réduit la fragmentation des membranes neutres et augmente la fragmentation des membranes anioniques. Malgré les interactions électrostatiques entre le peptide cationique et les lipides anioniques, aucune spécificité lipidique n'est observée dans l'extraction. La BSP1 est la protéine la plus abondante du liquide séminal bovin et constitue un autre exemple de PEL naturel important. Elle se mélange aux spermatozoïdes lors de l’éjaculation et extrait des lipides de leur membrane, notamment le cholestérol et les phosphatidylcholines. Cette étape cruciale modifie la composition lipidique de la membrane du spermatozoïde, ce qui faciliterait par la suite la fécondation de l’ovule. Cependant, le contact prolongé de la protéine avec les spermatozoïdes endommagerait la semence. Cette thèse cherche donc à approfondir notre compréhension de ce délicat phénomène en étudiant le mécanisme moléculaire par lequel la protéine fragmente les membranes lipidiques. Les résultats des présents travaux permettent de proposer un mécanisme d’extraction lipidique en 3 étapes : 1) L'association à l’interface des membranes; 2) La relocalisation de l’interface vers le cœur lipidique; 3) La fragmentation des membranes. La BSP1 se lie directement à deux PC à l'interface; une quantité suffisante de PC dans les membranes est nécessaire pour permettre l'association et la fragmentation. Cette liaison spécifique ne mène généralement pas à une extraction lipidique sélective. L'impact des insaturations des chaînes lipidiques, de la présence de lysophosphatidylcholines, de phosphatidyléthanolamine, de cholestérol et de lipides anioniques est également évalué. Les présentes observations soulignent la complexe relation entre l'affinité d'un PEL pour une membrane et le niveau de fragmentation qu'il induit. L'importance de la relocalisation des PEL de l'interface vers le cœur hydrophobe des membranes pour permettre leur fragmentation est réitérée. Cette fragmentation semble s'accompagner d'une extraction lipidique préférentielle seulement lorsqu'une séparation de phase est induite au niveau de la membrane, nonobstant les interactions spécifiques PEL-lipide. Les prévalences des structures amphiphiles chez certains PEL, ainsi que de la fragmentation en auto-assemblages discoïdaux sont discutées. Finalement, le rôle des interactions électrostatiques entre les peptides antimicrobiens cationiques et les membranes bactériennes anioniques est nuancé : les résidus chargés diminueraient l'association des peptides aux membranes neutres suite à l'augmentation de leur énergie de solvatation. / Lipid-extracting peptides and proteins (LEPs) bind to lipid membranes, extract lipids in the form of smaller auto-assemblies, and ultimately fragment membranes. In nature, this lipid extraction occurs in many different cell systems and causes various consequences, such as a modification of the membrane lipid composition or the cell death. This thesis focuses on the lipid extraction, or fragmentation, induced by the peptide melittin and the protein Binder-of-SPerm 1 (BSP1) on model lipid membranes. To this end, liposomes of different composition are prepared and incubated with melittin or BSP1. The association to membranes is determined by the LEPs intrinsic fluorescence, while the extraction is characterized by a combination of colorimetric phosphorus assays and liquid chromatography-mass spectrometry analyses (LCMS). Melittin is a cationic antimicrobial peptide, a very common category of LEP found in living organisms. Cationic antimicrobial peptides are interesting to medicine because they directly target membrane lipids. The action of many of these peptides is described by the carpet-like mechanism, by which they adsorb to membrane surface, induce the formation of pores and then cause the fragmentation of the membranes. In this thesis, melittin is used as a model peptide in order to study the mechanism by which cationic antimicrobial peptides fragment lipid membranes. Results show that the phosphocholine (PC) membrane fragmentation is reduced by a gradual demethylation of the ammonium group. Analysis of the fragmented material reveals that PC are preferentially extracted from membranes, due to a local enrichment in PC near melittin in the membrane. Furthermore, a melittin analogue, for which a majority of its cationic residues were neutralized, is used to investigate the role of the cationic character of native melittin. The neutralization increases the peptide affinity for neutral and anionic membranes, reduces fragmentation of neutral membranes and increases fragmentation of anionic membranes. Despite electrostatic interactions between the cationic peptide and the anionic lipids, no lipid specificity is observed in the extraction. BSP1 is the most abundant protein of the bovine seminal plasma and constitutes another example of important LEP found in nature. Upon ejaculation, it mixes with spermatozoa and extracts membrane lipids, such as cholesterol and phosphatidylcholines. This crucial process modulates the lipid composition of sperm membranes, which would then facilitate egg fertilization. However, a prolonged contact between the protein and spermatozoa could damage the semen. This thesis is looking to deepen our understanding of this delicate phenomenon by studying the molecular mechanism by which this protein fragments lipid membranes. Results of the present work suggest a 3-step mechanism for the extraction: 1) Association to membrane interface; 2) Relocation towards the lipid core; 3) Fragmentation of membranes. BSP1 binds directly to two interfacial PC; a sufficient quantity of PC in membranes is necessary for protein association and fragmentation. This specific binding generally does not lead to specificity in the lipid extraction. The impact of unsaturation of the lipid chains, of the presence of lysophosphatidylcholines, of phosphatidylethanolamines, of cholesterol and of anionic lipids is also studied. The present observations underline the complex relationship between a LEP affinity for membranes and the level of fragmentation it induces. The importance of LEP relocation, from the interface to the hydrophobic core of the membranes, for fragmentation is reiterated. This fragmentation seems to be lipid specific only when a phase separation of the lipids occurs in the membrane, notwithstanding specific LEP-lipid interactions. The prevalence of amphipathic structures in certain LEPs, as well as of the auto-assembled discoidal structures resulting from fragmentation is discussed. Finally, the role of electrostatic interactions between cationic antimicrobial peptides and anionic bacterial membranes is detailed: charged residues lower peptide association to neutral membrane due to an increase of their free energy of solvation.

Microalgae are a diverse group of oxygenic photosynthetic microorganisms which show great promise as a source of biofuel. However, significant challenges still remain before microalgae can be considered a viable source of biofuel. The main current challenges are nutrient sourcing and recycling as well as downstream processing. The algal cell wall and especially the presence of an algaenan cell wall in some Chlorophyte algae could be an important variable in determining downstream processing costs but not much comparative research has been done to elucidate this. The first part of the present study focuses on the recently isolated alga Pseudochoricystis ellipsoidea (Trebouxiophyceae) and its improvement and assessment for biofuel production. Random mutagenesis and FACS screening protocols were developed for the isolation of pigment and cell wall mutants but despite considerable efforts no suitable mutants could be identified in the first half of this project. Two 500 L raceway ponds as well as an algal growth room and bubble column bioreactors were set up to facilitate algal research at the University of Bath and assess the performance of P. ellipsoidea in realistic culture conditions. P. ellipsoidea showed a maximum growth of 1.53 divisions day-1 in semi-open raceway ponds, resistance to contamination and a 30% lipid content, making it particularly suitable for raceway pond cultures. In the second part of this project six species of Chlorophyte (“green”) algae, three of which produced algaenan, were compared for suitability to growth in anaerobic digestate and municipal wastewater as well as cell wall strength, permeability and suitability to hydrothermal liquefaction. We found that anaerobic digestate was a good medium for the growth of all species independently of autoclaving and that non-autoclaved wastewater was a very challenging medium. Algaenan production did not affect cell disruption by ultrasonication but growth stage and cell wall thickness did. Lipid extraction kinetics by chloroform/methanol were greatly affected by algaenan, meaning that this material is relatively impermeable to organic solvents. Cell wall thickness, cell volume and lipid content also had an effect on lipid extraction kinetics but this was only measurable after 180 minutes of extraction. 8 Hydrothermal liquefaction showed high solid and low oil yields, very low sulphur (≤0.1 %) as well as a 1.1 % -1.8 % nitrogen content which is significantly lower than most algal HTL studies to date. This suggests that stationary stage algae are more difficult to process but give a cleaner biocrude and reduce the loss of nitrogen through incorporation in the oil. Significant opportunities for optimisation still exist in the HTL process.