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Title: Production and characterisation of cocoa butter equivalents from high oleic sunflower oil by enzymatic rearrangement
Authors: Ray, Joydeep
Issue Date: 2013
Publisher: © Joydeep Ray
Abstract: Cocoa Butter (CB) is one of the most expensive and widely used raw materials in the chocolate and confectionery industries. Due to the rise in the cost of CB in recent years, the variability in quality and the uncertainty in supply, the development of speciality fats with similar triacylglycerol (TAG) compositions to CB with full compatibility has been encouraged. Desirable TAG compositions in a fat can be efficiently achieved via rearrangement of the TAG structure of liquid vegetable oils with selected fatty acids by lipase-catalysed reactions. The main aim of the work presented in this thesis is to produce and formulate different Cocoa Butter Equivalents (CBEs) via enzymatic acidolysis of High Oleic Sunflower Oil (HOSO). This was carried out using a 1,3 regiospecific lipase (Rhizopus oryzae) with 11 different stearic-palmitic acid mixtures at an oil:acid ratio of 1:1.3 (w/w), to produce specialised fats with a high disaturated monounsaturated TAGs (Sat-O-Sat). Diacylglycerols (DAG) were also formed (~7-10%) as a by-product. The TAG and DAG compositions of the acidolysed fat samples were measured using High Resolution Gas Chromatography (HR-GC) and High Performance Liquid Chromatography (HPLC). A probability model was developed which efficiently predicted the final conversions of the major TAGs in the reactions. The variation of TAG composition with time was used to study the reaction kinetics using two different reaction schemes including all possible acidolysis reactions of TAGs with Stearic, Palmitic and Oleic acids at the 1 and 3 TAG positions and as well as reactions involving DAGs with different rate constant assumptions. The later scheme with equal rate constants for equivalent reactions of palmitic and stearic acid produced the best fits for this particular reaction system. Hard stearin fractions with high StOSt content were then isolated from the acidolysed HOSO via removal of free fatty acids by short path distillation (SPD) followed by single stage acetone fractionation. The HOSO hard stearin samples contained varying amounts of StOSt (69-84%) and POSt (4.5-21%), and with overall Sat-O-Sat levels of approximately 90%. This is comparable with commercial Shea stearin which is widely used in CBE formulations. The crystallisation and polymorphic behaviour of the HOSO stearins was studied and compared with Shea stearin. However, the HOSO stearin fractions contained significant amounts of oxidised glycerides (OX) and DAGs particularly of 1,3-StO and so further comparisons were performed on samples which were subjected to an additional silica treatment to remove these minor components. A multi-methodological approach was used for characterization of the samples which included Differential Scanning Calorimetry (DSC) melting, isothermal crystallisation and stop-and-return experiments, X-Ray Diffraction (XRD), pulsed Nuclear Magnetic Resonance (p-NMR), Polarised Light Microscopy (PLM) and Raman microscopy. Four major polymorphs previously found with pure StOSt, α, γ, β , and β were detected in the HOSO stearin and Shea stearin samples. An evidence for a fifth (δ) polymorph was found in DSC experiments, but not via XRD or Raman spectroscopy. The effect of varying StOSt and POSt content in the HOSO stearins was significant, in that crystallisation (at 20°C) speeded up as the StOSt level increased and the POSt level decreased, The main crystallisation event was generally faster for samples with higher StOSt levels (lower POSt levels). The removal of minor components via silica treatment generally speeded transformations to higher polymorphs (γ, β and β). This was consistent with higher SFC values (similar to Shea stearin). The results suggested that the silica treated HOSO stearins could potentially replace Shea stearin in CBE formulations. CBEs were then formulated by blending the HOSO stearins with Palm oil Mid Fraction (PMF) maintaining a constant StOSt level (~30%) (with varying amounts of POSt and POP). These were compared with CB and standard CBEs (formulated using both untreated and silica treated shea stearins) in terms of composition, crystallisation, polymorphism and compatibility. The CBE blends displayed three major polymorphs, α, β and β when crystallised isothermally at 20°C or crystallised non-isothermally and remelted. In contrast, transformation to the higher polymorph (β polymorph) was not observed for CB. The main isothermal crystallisation event was generally faster for CBE blends with higher POSt levels (lower POP levels). The CBE blends with higher POP levels as well as trisaturated TAGs tended to transform faster to the β polymorph, though complete transformation was not achieved even after 7 days of storage at 20°C. The overall SFC values of the CBE blends were also generally higher than CB. Mixtures of CBEs with CB (85:15) and Butter fat (BU) (85:15:15) in similar proportions to the fat phase of dark and milk chocolate showed similar crystallisation and melting behaviour to pure CB. Microstructures and XRD patterns of the mixtures of the CBEs with CB and as well as BU, showed evidence of the β polymorph similar to CB after 7 days of storage at 20°C. Iso-solid phase diagrams of mixtures of CBEs with CB in different proportions showed satisfactory compatibility with no eutectic behaviour. Thus the CBEs formulated from the HOSO stearins and PMF can be successfully used to replace CB partially in confectionery applications. Finally, the isothermal crystallisation of CB and selected CBE blends was studied by Hot Stage Microscopy in conjunction with image analysis using a novel image processing algorithm. These were compared with experiments with pure StOSt, POSt and POP and their ternary mixtures. It was possible to visualise and analyse the entire growth history of individual crystals (nucleation and growth kinetics) of these fat samples at different temperatures and polymorphic forms. Nucleation and growth rates of the CB β crystals were found to vary approximately exponentially with decreasing temperature. The growth of β crystals could be studied by first generating small β crystals using melt mediated transformation from the β form. These showed constant growth rates. In some cases, the nucleation and growth rates of the Sat-O-Sat crystals were found to increase initially and then decrease with increasing isothermal temperature, due to the impingement effects from the other polymorphs. The Ternary system displayed shorter induction time and faster crystal growth than that of the naturally derived fat samples, presumably due to the absence of other components. The final solid fat content (crystallised fraction) of all the fat samples was found to decrease with increasing temperatures as expected in a fat system.
Description: A Doctoral Thesis. Submitted in partial fulfilment of the requirements for the award of Doctor of Philosophy of Loughborough University.
Sponsor: Loughborough University and IOI Loders Croklaan Europe
URI: https://dspace.lboro.ac.uk/2134/13880
Appears in Collections:PhD Theses (Chemical Engineering)

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