Bridging wax ester crystallization and wax-based oleogels

Semi-solid fat phases are functional food ingredients with a high nutritional value. They are decisive for the desired quality of various foods, by providing emulsion stability and plasticity in spreads, stabilizing air bubbles in ice cream or determining mouthfeel by controlling melting and release of flavors. This characteristic functionality results from a three-dimensional scaffolding in liquid oil, typically formed by triacylglycerides (TAGs) with a high content of saturated fatty acids (FA). The intake of these is associated with ecological, ethical, and health concerns and should therefore be reduced in the human diet. The field of oleogelation seeks alternative ways to structure liquid oil, using non-TAG molecules which are able to structure liquid oil. Among the various possible oleogelators, natural waxes fulfill most requirements, rendering them promising candidates as structuring agents. Inevitable key features for an oleogelator include high structure efficiency, approval as food ingredient, high potential consumer acceptance, or sustainable production possibilities. The term “natural waxes” describes high-melting, hydrophobic lipids from various animal or plant origin. Typical examples studied for food applications are beeswax (BWX), candelilla (CLX), carnauba (CRX), rice bran (RBX) or sunflower wax (SFX). Main constituents are long-chain aliphatic chain molecules – FAs, fatty alcohols (FaOHs), n-alkanes (HCs), and wax esters (WEs) – in varying fractions depending on the biological origin of the wax. SFX and RBX contain more than 95 wt.% WEs, making them very homogeneous in composition. Despite containing WEs as most prominent substance class, CRX and BWX contain higher amount of free FaOHs (CRX), FAs (CRX, BWX) and HCs (BWX). CLX, in contrast, is composed of 40-80 wt.% of HCs. Even though waxes have been subject to research for almost two decades until now, industrial product applications are still lacking. One limitation for product application is the current legislation, approving waxes only as coating or glazing agents for food applications and not as bulk phase ingredients. However, among others, functional issues such as brittle gel structure, sensory disadvantages like a sandy mouthfeel arising from high melting points and large crystalline structures but also inconsistent functionality of different waxes, hinder product implementation. The fact that no sufficiently profound knowledge exists about the relationship between composition, formed structure, and the resulting functionality for wax-based oleogels is a main reason. From an academic perspective, comprehensive summary of existing studies is hampered by a lack of comparability, as different oil types and qualities as well as distinct processing and analytical parameters significantly impact the results and their interpretation. Further, the specific molecular composition of the studied waxes is typically unknown. The aim of the work documented in this thesis was to bridge fundamental knowledge on WEs and macroscopic properties of wax-based oleogels to provide evidence-based groundwork for future application of natural waxes in oleogels. Therefore, these issues were addressed from two contrasting perspectives: Using a bottom-up approach, the first part investigated the crystallization and gelation behavior of pure WEs and oleogels formed therewith. The second part, in contrast, employed a top-down approach to study the molecular composition of several natural waxes, including batch-to-batch-variability, and the implications that can be derived for the network structure and macroscopic properties. Together, these two perspectives are designed to connect molecular-scale crystallization phenomena with macroscopic gel properties in real wax systems. The comprehensive elucidation of WE crystallization is considered a prerequisite to understanding wax-based oleogelation. To identify the molecular parameters governing wax-based oleogelation, the first part of this thesis systematically elucidated the crystallographic and thermal behavior of pure WEs. In particular, the two variable structural features – total carbon number (CN) and position of the ester bond within the alkyl chain (ΔCN) – were emphasized. Pure component WEs were characterized regarding their crystallographic and thermal behavior, using established analytical methods, X-ray scattering and differential scanning calorimetry (DSC) at standardized conditions. While subcell structure was found to be independent of molecular make-up, ΔCN was identified as decisive parameter for both lamellar stacking and thermal properties. In particular, only WEs with a FaOH moiety of 2 carbon atoms longer or 4 carbon atoms shorter than the FA moiety, exhibited an orthogonal chain orientation. This was accompanied by increased values of their thermal properties compared to their related homologues at equal CN. A predictive model for the enthalpy of fusion and the melting point temperature of WEs was required to extend the experimental findings. The availability of physical sample material is limited to CN52, which is below the expected CN range for natural waxes up to CN64. This necessity of obtaining the thermal properties of WEs without available experimental data, was satisfied by the group contribution approach chosen. According to their ΔCN, WEs were grouped into four categories. Established thermodynamic fundamentals for the melting point temperature and the enthalpy of fusion were confirmed for WEs and applied to predict WE properties. Studies on oleogels were performed to assess whether molecular insights from pure WEs translate into structured oil systems. By investigating gels made of single WEs in medium-chain triglyceride (MCT) oil WE-related effects were focused, excluding variable oil phase composition. The crystallographic and thermal properties of pure WEs are transferable from pure components to oleogels, when taking effects of solubility into account. Regarding the ideality of the WE solubility in MCT oil, the results of this work gave contrasting results. Based on the interpretation of experimental data, an increased solubility of WEs in MCT oil was suggested. The molecular dynamics simulation on model systems of simplified composition, however, indicated ideal behavior for short chain WEs and decreased solubility of long chain WEs due to decreased compatibility of WE and TAG chains. The formation of a crystal network ultimately determines oleogel rigidity and functionality beyond crystallographic properties. To study the network structure, microscopic techniques (light microscopy and cryo-scanning electron microscopy) as well as oscillatory rheology were applied. While all mixtures of pure WEs exhibited platelet-like crystal morphology, natural waxes formed less regular, card-house like or aggregated networks. Increasing WE chain length led to more regular platelet morphologies with distinct crystallographic habits, which directly affected gel rigidity. However, it was found that the microstructure and gel rigidity were more affected by the gel formation kinetics than by the molecular makeup, regardless of whether the reduction in crystal size and homogeneity was due to a higher cooling rate or variations in molecular structure. Since natural waxes represent complex mixtures of WEs, understanding the crystallization behavior of WE mixtures is essential for predicting real wax performance. The mixing behavior of WEs in oleogels was investigated using ternary mixtures of two WEs in MCT-oil. For a mechanistic understanding, the chain length disparity between the WEs was considered additionally to ΔCN. While high chain length disparities yielded sequential crystallization, medium chain length disparities exhibited several more solid phases than WEs present in the mixture. It has been found that the lower-melting phase utilizes the existing network of the higher-melting phases as a growth site, resulting in increased gel rigidities for such systems. This is indicative of sintering, the formation of secondary bonds that reinforce the crystal network. For small chain length disparities, no clear differences in thermal behavior or network properties were identified. While the first part established WEs as the primary structural and thermodynamic determinants of wax‑based oleogels, these model systems cannot capture the full compositional complexity of natural waxes. Consequently, profound knowledge of the specific molecular composition, linked to the functional macroscopic properties of natural waxes provided the rationale for the second part of the thesis. A prerequisite for linking natural wax composition to functionality is the availability of an analytical method capable of resolving their major aliphatic constituents. Compositional analysis of natural waxes requires an easily applicable analytical method sensitive to the aliphatic major constituents. The development of a novel LC-ELSD method enabled simultaneous analysis of four substance classes of underivatized samples at ambient temperatures. Substance-specific response factors were obtained from a multi-level calibration with external standards. These factors were employed to obtain relative compositions by normalization. Comparison of the analysis results with both literature and high temperature GC data confirmed the LC results, proving the method’s validity. Not much is known about the selectivity of WE biosynthesis for specific combinations of FAs and FaOHs. Using FTIR, LC-ELSD and GC-FID as complementary techniques, the analysis of native and partially hydrolyzed waxes confirmed the hypothesis that CN profiles largely result from statistical recombination of available FAs and FaOHs. Based on this, general patterns for WE composition in natural waxes including ΔCN patterns were derived. It was found that the WE diversity is higher for SFX than for RBX and lowest for BWX. Overall, this study revealed the presence of highly