Enzymic Interesterification

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). 

Lipases can be classified into groups that reflect their specificity. The common lipases include non-specific lipases that do not discriminate between the position or the type of the fatty acid on the triacylglycerol (e.g., lipase from Candida cylindracea) and 1,3-specific lipases that act only at the sn-l and sn-3 positions of the triacylglycerol (e.g., lipases from Aspergillus niger and Rhizopus species). In addition, some lipases are specific for a specific fatty acid type (e.g., lipase from Geotrichum candidum). 

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. 

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Enzymic Interesterification of Milk Fat with Free Fatty Acids... 

Enzyme interesterification is rapidly becoming popular and has the advantage of selecton of TAG positions at which fatty acids are interchanged. When 1,3 lipases are used, the... 

Bohenin (BOB) is the name given to glycerol 1,3-behenate 2-oleate, which inhibits fat bloom when added to chocolate. It is produced in Japan by enzymic interesterification of triolein and behenic (22 0) acid or ester in the presence of a 1,3 stereospecific lipase. 

Wisdom, R. A., Dunnill, R, and Lilly, M. D., Enzymic interesterification of fats the effect of non-lipase material on immobilized a.zyme activity. Enzyme Microb. Tech-nol, 71, 567-572, 1985. 

Enzymic interesterification is a recent development of considerable promise. Its advantage over the more conventional procedures lies in the additional control of product composition. The lipase, coated on to a support material (kieselguhr, hydroxylapatite, alumina) in the presence of a little water, supports interesterification at about 40 °C and usually requires 16-70 h for complete reaction but can be shorter depending on catalyst activity. The process may be operated in a batch or continuous manner. The substrate is a natural oil or fat to which may be added a second oil or fat and/or a particular fatty acid or fatty acid mixture. Three types of enzyme are available. One type (from Candida cylindricae, Coryne-bacterium acnes or Staphylococcus aureus) is nonspecific and leads to complete randomization of all acids at all positions. The product is the same as that obtained from the ordinary catalytic process. A second type of lipase (from Aspergillus niger, Mucor favanicus, Rhizopus arrhizus, / . delemar... 

Wisdom, R.A., Dunnill, P. and Lilly, M.D. (1987) Enzymic interesterification of facts. Laboratory and pilot scale studies with immobilized lipase from Rhizopus arrhizus. Biotechnol. Bioeng. 29, 1081-1085. 

Z Lin, A Qiu, X Wang. Advances of enzymic interesterification in supercritical carbon dioxide. Zhongguo Youzhi 22(2) 26-28, 1997. 

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. 

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 dedd cells of the mycelium of Rhizopus arrhizus constitute d naturally immobilized lipdse very active in organic solvents. This immobilized enzyme was used for hydrolysis and synthesis of ester bonds triglycerides hydrolysis, and interesterification, esters and glycerides synthesis. More recently, the catalytic system has been applied in drug synthesis to the resolution of racemic esters with a good enantioselectivity. 

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. 

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. 

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],... 

One of the limitations encountered with the use of physical blends of different fats is incompatibility of the fats causing softening of fats due to eutectic effects. Nor Hayati et al. (2000) demonstrated that the eutectic effects observed in physical blends of milk fat and palm stearin was reduced on interesterification of the blend by a 1,3-specific enzyme (Lipozyme). The interesterified blend has better functionality for bakery products than milk fat. 

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). 

Enzymes have been used to assist oil extraction and in degumming (phosphatides removal), splitting fatty acids from triglycerides, interesterification (rearranging fatty acids on triglyceride molecules), and preparation of specialty oils. These processes are described later in this chapter. 

Fig. 34.30. Schematic drawing of multiple enzyme reactor system for enzymatic interesterification of trans-free margarine and shortening oils. (Courtesy of Novozymes A. S, Bagsvaerd, Denmark.)...

The thermostability of the immobilized lipase is most easily demonstrated in continuous interesterification. A general test system is shewn in Figure 6. The immobilized enzyme is placed in the enzyme column. The precolumn is used to saturate the reactants with water as they are pumped through the system. 

The activities in acidolysis of tricaprin with different fatty acids were measured in separate experiments, each performed like the assay for batch interesterification units. It is observed that the enzyme has seme specificity towards saturated fatty acids. This kind of specificity is unique, and has not previously been described. 

Another promising area for adaptation of enzyme bioreactor technology is that of lipid modification. Several examples are a) the interesterification of triacylglycerols to change their composition b) limited lipolysis for production of flavors and c) conversion of cholesterol to forms that are not absorbed. The potential stabilization of enzymes to the presence of organic solvents would provide a definite advantage to enzyme bioreactor technology for the modification of lipid molecules. 

Bohenin occurs as a white to light tan, waxy solid. It is a triglyceride containing behenic acid at the 1- and 3-positions and oleic acid at the 2-position. Behenic acid is a saturated fatty acid that occurs naturally in peanuts, most seed fats, animal milk fat, and marine oils. It is produced by the interesterification of triolein and ethyl behenate in the presence of a suitable lipase enzyme preparation. It melts at approximately 52°. It is insoluble in water soluble in hexane, in chloroform, and in acetone and slightly soluble in hot ethanol. 

Lipase catalyzed reactions take place in the neat oil or in a nonpolar (usually hydrocarbon) solvent. The efficiency depends on the amount of water, solvent (if present), temperature, and ratio of reactants. A factorial approach can be used to optimize the conditions (32). In interesterification reactions, 1,3-specific enzymes give control over product composition that is not possible using chemical catalysts. For example, starting with SOS and OOO, chemical interesterification produces aU eight possible isomers (see Table 5). Enzymatic interesterification does not exchange fatty acids at the sn-2 position, and it will result in only two additional molecular species, OOS and SOO. In more realistic situations, chemical and enzymatic interesterification may produce the same or a similar number of molecular species, but in different proportions (31). 

Considerable recent research has defined conditions for successful use of lipases and other enzymes in numerous lipid modification reactions, including a variety of types of interesterifications (69, 71, 76). For edible applications to date, they have been employed at industrial scales for the production of (1) cocoa butter substitutes, for which disaturated, monounsaturated acylglycerols with the unsaturated fatty acid in the sn-2 position are desired (77) (2) to produce human milkfat analogues, where 2-palmitoyl acylglycerols are desired (77) (3) in the synthesis of 1,3- di-acylglycerols (78) and in the production of diacylglycerols for edible applications. These reactions employ vegetable oils as feedstocks. 

The production of fat spreads as an alternative to butter led to an increased demand for solid fats. For the most part, this demand has been met by the use of partially hydrogenated vegetable oUs (Section 8.3), but concern about the health effects of trani-unsaturated acids has raised interest in an alternative way of producing fats with the required melting behavior. This can be achieved by interesterification of blends of natural or fractionated fats. Products obtained in this way will probably contain more saturated acids than their partially hydrogenated equivalents, but they will have no trans-acids. This section is devoted to interesterification carried out under the influence of a chemical catalyst (177, 186, 187). Similar reactions with enzymes are discussed in the following section. 

Natural oils and fractionated oils usually have their acyl chains organized in a nonrandom manner, but they become randomized after interesterification with a chemical catalyst. There is no change in fatty acid composition, only in triacylglycerol composition, but this leads to a modification of the physical properties. More selective interesterification can be achieved with enzymic catalysts (Section 8.5). 

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