Lipases interesterification

Lipase. Interesterification of triacylglycerols in the presence of a regiospecific lipase or a nonspecific lipase and free fatty acids can be used to modify the physical or nutritional characteristics of fats. Lipases from Aspergillus sp. (47,48) and Rhizopus... 

Papaya latex lipase Interesterification of tributyrin and Foglia and Valivety... 

In addition to having the required spedfidty, lipases employed as catalysts for modification of triglycerides must be stable and active under the reaction conditions used. Lipases are usually attached to supports (ie they are immobilised). Catalyst activity and stability depend, therefore, not only on the lipase, but also the support used for its immobilisation. Interesterification reactions are generally run at temperatures up to 70°C with low water availability. Fortunately many immobilised lipases are active and resistant to heat inactivation under conditions of low water availability, but they can be susceptible to inactivation by minor components in oils and fats. If possible, lipases resistant to this type of poisoning should be selected for commercial operations. 

Lipases catalyse reactions at interfaces, and to obtain a high rate of interesterification the reaction systems should have a large area of interface between the water immiscible reactant phase and the more hydrophilic phase which contains the lipase. This can be achieved by supporting the lipase on the surface of macroporous particles. 

The role of reversed micelles in the manufacture of fine chemicals with enzymes also needs to be assessed and analysed. An outstanding example is lipase catalysed interesterification to produce cocoa butter substitute from readily available cheap materials (Luisi, 1985). This example of reversed micelles is sometimes referred to as a colloidal solution of water in organic systems. A number of water insoluble alkaloids, prostanoids, and steroids have been subjected to useful transformations (Martinek et al., 1987). Peptide synthesis has also been conducted. The advantages of two liquid phases are retained to a very great extent the amount of water can be manipulated to gain advantages from an equilibrium viewpoint. 

Fixed-bed reactors employed for lipase-catalyzed hydrolysis and interesterification reactions are highly efficient and have been used on a large scale (Table 5). The two phases may flow through the reactor in the opposite or same directions. If no solvents are used, the effect of viscosity of some substrates (i.e., oil) may be minimized by employing high temperatures which lead to faster rates of inactivation of lipases. 

At present, margarine producers are moving to use fractionation and interesterification to produce the required properties. A new technology uses lipase enzymes to rearrange fatty acids in a controlled way. 

An alternative method is interesterification where the fatty acids are rearranged. This can be done chemically, which gives a random distribution, or by using enzymes. The advantage of enzymes is that they are very specific in their action. It is quite possible using a lipase to remove... 

Natural fats and oils can be used directly in products, either individually or as mixtures. In many cases, however, it is necessary to modify their properties, particularly their melting characteristics, to make them suitable for particular applications. Therefore, the oils and fats industry has developed several modification processes using enzyme technology. In particular, lipases (and lately cutinases), phospholipases and pectinases can be used for interesterification processes, ester syntheses and in olive-oil extraction. 

The main current potential application of lipase-catalyzed fat-modification processes is in the production of valuable confectionery fats for instance, alternative methods of obtaining cocoa-butter equivalents by converting cheap palm-oil fats and stearic acid to cocoa-butter-like fats. The reaction is executed in a water-poor medium, such as hexane, to prevent hydrolysis. At least one commercial apphcation exists. Loders Croklaan (Unilever) has an enzymatic interesterification plant in Wormerveer, the Netherlands. Many other new potential applications of lipases have been proposed of which some will certainly be economically feasible. Examples and details can be found in chapter 9 of this book. 

Lipases are used to hydrolyse milk fat for a variety of uses in the confectionary, sweet, chocolate, sauce and snack food industries and there is interest in using immobilized lipases to modify fat flavours for such applications (Kilara, 1985). Enzymatic interesterification of milk lipids to modify rheological properties is also feasible. 

The interesterification of fats and oils is the only way to create new hybrid products with new physical, and especially new rheological properties. Chemical interesterification is well known, but has no position or chain specificity, and is not very clean. With lipases in micro-aqueous media, the exchange of acyl groups between the different triglycerides may be oriented, and designed according to the specificity of the enzyme. 

Rhizopus arrhizus dead mycelium was found to be very active in organic solvents as a naturally immobilized lipase. Triglycerides hydrolysis and interesterification, esters and glycerides synthesis, natural flavour esters preparation and racemic mixtures resolution in pharmaceutical drugs synthesis are among the successfully designed processes, each of one with a specific reactional medium. 

Michor, H. Marr, R. Gamse, T. Schilling, T. Klingsbichel, E. Schwab, H. Enzymatic Catalysis in Supercritical Carbon Dioxide Comparison of Different Lipases and a Novel Esterase. Biotechnol. Lett. 1996b, 18, 79-84. Miller, D. A. Blanch, H. W. Prausnitz, J. M. Enzyme-Catalyzed Interesterification of Triglycerides in Supercritical Carbon Dioxide. Ind. Eng. Chem. Res. 1991, 30, 939-946. 

Yoon, S. H. Nakaya, H. Ito, O. Miyawaki, O. Park, K. H. Nakamura, K. Effects of Substrate Solubility in Interesterification with Riolein by Immobilized Lipase in Supercritical Carbon Dioxide. Biosci. Biotechnol. Biochem. 1998, 62, 170-172. Yu, Z. R. Rizvi, S. S. H. Zollweg, J. A. Enzymatic Esterification of Fatty Acid Mixtures from Milk Fat and Anhydrous Milk Fat with Canola Oil in Supercritical Carbon Dioxide. Biotechnol. Prog. 1992, 8, 508-513. 

Recent studies in the pharmaceutical field using MBR technology are related to optical resolution of racemic mixtures or esters synthesis. The kinetic resolution of (R,S)-naproxen methyl esters to produce (S)-naproxen in emulsion enzyme membrane reactors (E-EMRs) where emulsion is produced by crossflow membrane emulsification [38, 39], and of racemic ibuprofen ester [40] were developed. The esters synthesis, like for example butyl laurate, by a covalent attachment of Candida antarctica lipase B (CALB) onto a ceramic support previously coated by polymers was recently described [41]. An enzymatic membrane reactor based on the immobilization of lipase on a ceramic support was used to perform interesterification between castor oil triglycerides and methyl oleate, reducing the viscosity of the substrate by injecting supercritical CO2 [42],... 

Kalo, P., Parviainen, P. Vaara, K., Ali-Yrrko, S., Antila, M. 1986a. Changes in the triglyceride composition of butter fat induced by lipase and sodium methoxide catalysed interesterification reactions. Milchwissenschaft. 41, 82-85. 

Kalo, P., Vaara, K., Antila, M. 1986b. Changes in triglyceride composition and melting properties of butter fat fraction/rapeseed oil mixtures induced by lipase catalysed interesterification. Fette, Seifen, Anstrichmittm. 88, 362-365. 

Kalo, P., Huorati, K. H., Antila, M. 1990. Pseudomonasfluorescens lipase-catalysed interesterification of butter fat in the absence of a solvent. Milchwissenschaft. 45, 212-285. 

Lipases may be used to lipolyse milk fat to produce dairy flavor enhancers or for interesterification of milk fat systems to produce milk fat with improved nutritional or physical properties. Lipases may be used with or without an organic solvent in the system (de Greyt and Huyghebaert, 1995 Rousseau and Marangoni, 2002). 

The action of the lipase, its stability and rate of reaction are influenced by many factors, including temperature, pH, type of solvent, water activity and whether it is in an immobilized or free form (Valivety et al., 1994 Soumanou et al., 1999 Ma et al., 2002, Rousseau and Marangoni, 2002). Liquid butteroil by itself can act as a solvent as well as a substrate and interesterification is enhanced in the presence of an organic solvent such as hexane (Lee and Swaisgood, 1997). 

Interesterification of milk fat has been carried out by various free and immobilized lipases in both solvent and solvent-free systems. 

Safari et al. (1993) examined the interesterification of milk fat by the lipase from Rhizomucor miehei in various organic solvents (hexane, hexane-choloroform (70 30, v/v), and hexane-ethylacetate (70 30, v/v)). The addition of chloroform or ethyl acetate to hexane increased lipase activity. It was suggested that the polarity of the solvent influences the partitioning of water in the system with consequent effects on enzymic activity. Bornaz et al. 

Interesterification of blends of milk fat and palm kernel olein by a mycelium-bound lipase from Rhizomucor miehei or a commercially immobilized enzyme preparation resulted in a lower slip melting point and solid fat content. An interesterified product made from a 70 30 mixture of palm kernel olein and anhydrous milk fat was considered to be suitable for use in ice cream (Liew et al., 2001). 

Similar changes in chemical composition and melting properties were reported for chemical and enzymatic interesterification of milk fat with a non-specific lipase (Kalo et al., 1986 a, b). Both processes result in randomisation of the fatty acids. 

When a 1,3-specific lipase is used for interesterification, the enzymatically-modified product has some different properties compared to those of a chemically-interesterified product. For example, the dropping point of butterfat was increased slightly by chemical interesterification whereas interesterification by a 1,3-lipase from Rhizopus arrhizus led to a 2-4°C decrease in dropping point. Although both methods of interesterification reduced hardness, the magnitude of the effect was greater for the enzymatically-interesterified fat (Marangoni and Rousseau, 1998). 

When 80 20 blends of butter fat and canola oil were used, chemical interesterification increased the solid fat content above 10°C while enzymatic interesterification by Rhizopus arrhizus lipase reduced solid fat content over the range 5 40 C (Rousseau and Marangoni, 1999). 

Balcao, V.M., Kemppinen, A., Malcata, F.X., Kalo, P.J. 1998a. Modification of butterfat by selective hydrolysis and interesterification by lipase process and product characterization.. / Am. Oil. Chem. Soc. 75, 1347-1358. 

Balcao, V.M., Malcata, F.X. 1998b. Interesterification and acidolysis of butterfat with oleic acid by Mucor javanicus lipase changes in the pool of fatty acid residues. Enz. Microb. Technol. 22, 511-519. 

Oba, T., Witholt, B. 1994. Interesterification of milk fat with oleic acid catalyzed by immobilized Rhizopus oryzae lipase. J. Dairy Sci. 77, 1790-1797. 

Rousseau, D., Marangoni, A.G. 1998a. Tailoring attributes of butter fat/canola oil blends via Rhizopus arrhizus lipase-catalyzed interesterification. 1. Compositional modifications. J. Agric. Food Chem. 46, 2368—2374. 

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