Enzymatic interesterification

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. 

Ison, A.P., Dunnill, P., Lilly, M.D., Macrae, A.R. and Smith, C.G. (1990) Enzymatic interesterification of fats Immobilization and immimogold localization of hpase on ion-exchange resins. Biocatalysis, 3, 329-342. 

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. 

Organic solvents used must be compatible with the enzymatic activity, not take place in the reaction, be good solvent of the substrates, have a low cost, and be the more harmless possible. The solvents commonly used in the literature are aliphatic alkanes, or similar compounds of low polarity. Aliphatic ethers that were used in this work, show less hydrophoblcity than alkanes, and thus allow introduction of water for hydrolysis reactions. For interesterification experiments, trichloro-trifluoro-ethane was used, as water is not a substrate for the reaction (Fig. 5,). 

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],... 

Ferrari, R., Esteves, W. and Mukherjee, K. (1997) Alteration of steryl ester content and positional distribution of fatty acids in triacylglycerols by chemical and enzymatic interesterification of plant oils. J. Am. Oil Chem. Soc., 74(2), 93—96. 

Garcia, H.S., Storkson, J.M., Pariza, M.W., Hill Jr., C.G. 1998. Enrichment of butteroil with conjugated linoleic acid via enzymatic interesterification (acidolysis) reactions. Biotechnol. Lett. 20, 393-395. 

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). 

Solvent-free enzymatic interesterification of milk fat alone or with other fats or fatty acids provides the most acceptable route for modification of the triacylglycerol structures in milk fat and further research and development in this field is expected to provide physical and physiological benefits. From a nutritional perspective, it is of interest to examine the effects of randomized milk fat on serum cholesterol. Christophe et al. (1978) reported that substitution of native milk fat with chemically-randomized interester-ified milk fat reduced cholesterol levels in man. However, others found that there was no effect on serum cholesterol levels in man as a result of substitution of ezymatically randomized milk fat (De Greyt and Huyghebaert, 1995). Further studies are required to determine if interesterilied milk fat provides a nutritional benefit. 

Nor Hayati, I., Aminah, A., Mamot, S., Nor Aini, I., Noor Lida, H.M., Sabariah, S. 2000. Melting characteristic and solid fat content of milk fat and palm stearin blends before and after enzymatic interesterification. J. Food Lipids. 7, 175-193. 

Interesterification (INES) is the exchange of acyl radicals between an ester and an acid (aci-dolysis), an ester and an alcohol (alcoholysis), or an ester and an ester (transesterification), and can be random, directed, or enzymatic. The process has been called intraesterification if an exchange of positions occurs within the same molecule, and randomization if exchange occurs between molecules.44,48 The principles can be used to position fatty acids on mole-... 

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.)...

Enzymatic reactions in organic media have been a major issue in the field of biocatalysis over the last two decades. Carboxylesterases (mostly lipases) have been used in monophasic organic solution under controlled values of water activity (ajj for catalyzing ester formation the reaction equilibrium can be shifted towards ester formation by interesterification or transesterification [1]. Direct esterification is often hampered by water formation, which may increase o , thus negatively influencing the equihbrium. 

Rousseau, D., and Marangoni, A.G. (1998). The effeets of chemical and enzymatic interesterification on the physical and sensory properties of butterfat-canola oil spreads. Food Research International. 31 381-388. 

Under almost anhydrous conditions in organic medium, lipases can be used in the reverse mode for direct ester synthesis from carboxylic acids and alcohols, as well as transesterifications (acyl transfer reactions) which can be divided into alcoholysis (ester and alcohol), acidolysis (ester and acid), and interesterification (ester-ester interchange). The direct esterification and alcoholysis in particular have been most frequently used in asymmetric transformations involving lipases. The parameters that influence enzymatic catalysis in organic solvents have been intensively studied and discussed. ... 

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). 

Interesterification by chemical or enzymatic catalyst Domestication of wild crops... 

Chemical interesterification is conveniently achieved by using alkali metal methylates as a catalyst. Microbial lipases are also used as biocatalysts in enzymatic interesteiification. In contrast to the chemical process, the enzymatic process can be more selective if an enzyme with positional specificity is used, but this reaction is usually much slower and more sensitive to reaction conditions. Recent developments in lipase-catalyzed interesterification have resulted in new industrial applications of this process (255). Nevertheless, the high costs of enzymes and process equipment may limit widespread adoption of this process. 

Microbial lipases may be used as biocatalysts for interesterification. Lipase-assisted interesterification offers possibilities for transformation of lipids beyond those possible using chemical interesterification (11). Enzymatic interesterihcation has several advantages over the chemical-assisted reactions, such as mild reaction conditions leading to reduced energy consumption and less thermal damage to reactants and products, the possibility of lipase specificity toward their natural substrates, as well as high catalytic efficiency. Lipase-catalyzed interesterihcation reactions, in contrast to those carried out with chemical catalysts, yield different types of products depending on the specihcity of the lipase used. 

Interesterification is done by two methods chemical and enzymatic. The cost for making interesterified producs is very similar for both processes. The enzymatic interesterification process is becoming more popular because it is environmentally friendly. 

A new technology, an alternative to chemical interesterification, namely enzymatic interesterification is emerging. The process was introduced in the United States in 2003. This process is patented by Novozymes. Figure 5 depicts this process in a simplified form. 

One must understand that the interesterification process, chemical or enzymatic, is of no value in making heavy-duty frying fats. The high percentage of unsaturated oil, even with interesterification and addition of near-zero IV fat to provide plasticity, is still unstable under heavy-stress frying conditions. 

SCFs offer a nonaqueous environment which can be desirable for enzymatic catalysis of lipophilic substrates. The lipophilic substance cholesterol is 2 to 3 orders of magnitude more soluble in CX>2-cosolvent blends than in waterQ). In CO2 based blends, it may be oxidized to cholest-4en-3one, a precursor for pharmaceutical production using an immobilized enzyim(22). The enzyme polyphenol oxidase has been found to be catalytically active in supercritical CO2 and fluoroform (22). The purpose of using a SCF is that it is miscible with one of the reactants-oxygen. Lipase may be used to catalyze the hydrolysis and interesterification of triglycerides in supercritical OO2 without severe loss of activity(24). These reactions could be integrated with SCF separations for product recovery. 

Structured lipids (SLs) are defined as TAGs restructured or modified to change their FA composition or their positional distribution in TAG molecules by chemical or enzymatic reaction, such as direct esterification, acidolysis, alcoholysis, or interesterification, depending on the types of substrates available (Lee and Akoh, 1998). 

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