Lipase Catalysed Modification of Animal Fats

Abstract

Chapter 1 gives a brief introduction to lipids, their nomenclature and physical properties. Some of the nutritional properties of fats and oils are outlined, including a discussion of the differences (physical and nutritional) between animal and vegetable fats. Some reasons for, and examples of, modifying animal fats by physical, chemical or enzymatic means are also discussed. In Chapter 2, chemicals, apparatus and instrumentation, and standard or routine analytical methodologies are described. A discussion of the method used for analysis of free fatty acid composition is extended in Appendix A. Chapter 3 describes a preliminary investigation into lipase-assisted fractionation of tallow. On a small scale (< 50 g of fat) immobilised lipases were used to catalyse interesterification of tallow triglycerides using a temperature at which crystals of highly saturated fat would form. The resulting oleins contained significantly higher levels of unsaturated fatty acids than were obtained in the absence of lipase. The effect was small when dry (solvent free) fractionation was used but was much more marked in the presence of 30% w/w acetone. The acetone allowed more vigorous agitation of the reaction mixture while maintaining the formation of well-defined, easily filtered stearin crystals. Reaction for 24 hours with 2% Novozym 435 gave modest increases in olein unsaturation (41-46%) but recycling of the olein 4 times yielded 56% unsaturation. Similar results could be obtained in a single-step process by increasing the reaction time to 14 days. Chapter 4 discusses scaling up of the lipase-directed fractionation technique. Melted tallow was circulated through a packed bed enzyme reactor and a separate crystallisation vessel. The temperatures of the two parts of the apparatus were controlled separately to allow crystallisation to occur separately from interesterification. Operation of the reactor with conventionally dry, pre-fractionated tallow allowed the formation of an olein consisting of up to 60% unsaturated fatty acids. The greatest changes in olein fatty acid composition were achieved when the fractionation temperature was kept constant at a value that promoted selective crystallisation of trisaturated triglycerides that were continuously produced by enzymic interesterification. The enzyme could be reused without apparent loss of activity and its activity was apparently enhanced by preincubation in melted tallow for up to several days. Control of both the water activity of the enzyme and tallow feedstock and the absorption of atmospheric water vapour were required to maintain enzyme activity and minimise free fatty acid formation. This method may form the basis for a process to produce highly mono-unsaturated tallow fractions for use in food applications (e.g. frying) where a "healthy" low saturated fat product is desired. Chapter 5 investigates aspects of pregastric lipase catalysed hydrolysis of milk fat. Using cream (unpasteurised or pasteurised) it was shown that the pregastric lipases have selectivities (positional or chain-length) quite distinct from that of a variety of other classes of lipases (e.g. microbial, pancreatic). There was also quite an apparent difference in selectivity between that of calf PGL and that of lamb, kid and goat PGLs. Using similar analysis of a large range of commercially available PGLs, differences in selectivity were shown to be inherent to the species of enzyme, and not greatly influenced by other factors (e.g. country of origin, feeding regime, breed). Temperature and pH effects on the activity and selectivity of PGLs in the catalysed hydrolysis of milk were also investigated. All PGLs investigated displayed highest activity at 40ºC. However, temperature effects on selectivity were more ambiguous. It is possible that seasonal variation in the substrate affected the results and this will require further investigation. Finally a quick survey of the FFA composition of a variety of cheeses demonstrated that hydrolysis profiles obtained for cheeses which had used PGLs in their manufacture give comparable results with those of the earlier PGL milk hydrolysis experiments. It was also evident that lipase activity was retained after the cheeses had been placed into storage. In Chapter 6, the viability of using lipases to remove myristic acid (a hypercholesterolemic fatty acid) from tallow triglycerides was investigated. None of the lipases investigated were specific to the removal of myristic acid. However, it was apparent that some lipases were more suited to this application than others. The potential of PGLs to catalyse interesterification of oils containing short chain fatty acids was also investigated by using blends of natural fats (coconut oil and cocoa butter) or synthetic triglycerides. It appears that these lipases are unsuited to this application. The positional selectivity of PGLs was investigated using a variety of natural fats. These fats (cocoa butter and lard) have a very structured (non-random) positional distribution of their fatty acids. By analysing the PGL catalysed hydrolysis products it was found that calf PGL is quite positionally non-selective while goat PGL and lamb PGL (to a lesser degree) are 1,3-regioselective. These findings were confirmed by the use of synthetic triglycerides.