Production of Cocoa Butter-Like Fats by the Lipase-Catalyzed Interesterification of Palm Oil and Hydrogenated Soybean Oil Roland D
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Production of Cocoa Butter-like Fats by the Lipase-Catalyzed Interesterification of Palm Oil and Hydrogenated Soybean Oil Roland D. Abigora,b, William N. Marmerb, Thomas A. Fogliab,*, Kerby C. Jonesb, Robert J. DiCicciob, Richard Ashbyb, and Patrick O. Uadiac aBiochemistry Division, Nigerian Institute for Oil Palm Research, Benin City, Nigeria, bUSDA, ARS, ERRC, Wyndmoor, Pennsylvania 19038, and cDepartment of Biochemistry, University of Benin, Benin City, Nigeria ABSTRACT: Cocoa butter-like fats were prepared from re- Numerous fats suitable for total or partial replacement of fined, bleached, and deodorized palm oil (RBD-PO) and fully cocoa butter in confectionery products have been identified. hydrogenated soybean oil (HSO) by enzymatic interesterifica- Palm oil is an important fat used in the production of cocoa tion at various weight ratios of substrates. The cocoa butter-like butter-like fats (7,8,11,12). There are few papers, however, fats were isolated from the crude interesterification mixture by regarding the production of cocoa butter-like fats from palm fractional crystallization from acetone. Analysis of these fat oils based on their geographical origin, which may affect the products by RP-HPLC in combination with ELSD or MS detec- content of important TAG such as palmitoyl-oleoyl-stearoyl tion showed that their TAG distributions were similar to that of cocoa butter but that they also contained MAG and DAG, glycerol(POS) (11) in the final product. This paper reports the which were removed by silica chromatography. The optimal production of cocoa butter-like fats by lipase-catalyzed inter- weight ratio of RBD-PO to HSO found to produce a fat product esterification of palm oil and fully hydrogenated soybean oil containing the major TAG component of cocoa butter, namely, (HSO) and compares the fatty acyl and TAG composition and 1(3)-palmitoyl-3(1)-stearoyl-2-monoolein (POS), was 1.6:1. The melting profile of these products to cocoa butter. m.p. of this purified product as determined by DSC was compa- rable to the m.p. of cocoa butter, and its yield was 45% based on the weight of the original substrates. MATERIALS AND METHODS Paper no. J10538 in JAOCS 80, 1193–1196 (December 2003). Materials. HSO (16% C16:0, 84% C18:0, iodine value <5) was a gift from Nabisco Brands (Indianapolis, IN). Nigerian palm KEY WORDS: Cocoa butter-type fats, interesterification, lipase, oil was refined, bleached, and deodorized (RBD-PO) at the palm oil, soy oil. Food Protein Research and Development Center (College Sta- tion, TX). Lipozyme IM (an immobilized preparation of a 1,3- Rhizomucor miehei World production of cocoa butter was about 675,000 metric positionally specific lipase from ) was a tons in 2001 (1). Cocoa butter is currently the fat of choice in product of Novozyme (Franklinton, NC). TAG standards were chocolate and other confectionery industries based on its im- purchased from Sigma (St. Louis, MO), and the cocoa butter portant organoleptic and physical properties in these applica- standard was a gift from Loders Croklaan (Channahon, IL). Methods. (i) Enzymatic interesterification. tions. Numerous factors, however, including the degree of un- RBD-PO and certainty in supply, variability in quality, and price premium HSO were blended at weight percent ratios of 3:1; 1.6:1; 1:1; compared to other fats, have driven the search for alterna- 1:1.6; and 1:3. Interesterification of the blends was carried out tives. Numerous reformulated vegetable oils are currently in screw-capped sealed vials fitted with a magnetic stirring produced as cocoa butter equivalents (CBE), with the poten- bar. The vials were placed into a water-jacketed beaker tial market for CBE being as much as 10% of the total cocoa through which thermostatted water was circulated. Reactions butter market (2,3). were carried out at 70°C for 4 h with magnetic stirring at 200 Attempts have been made to prepare cocoa butter-like fats rpm. The reaction mixtures (3 g, total substrate mixture) were by interesterification of hydrogenated cottonseed oil and olive heated for 5 min at 70°C before addition of the lipase (10 wt% oil and subsequent fractionation (4). Edible beef tallow also of substrates). The optimal weight ratio of substrates (blend) has been solvent-fractionated from acetone to produce cocoa was ascertained by determining the production of the major butter-like fractions (5). The preparation of cocoa butter sub- TAG component of cocoa butter, namely, POS. (ii) Isolation of cocoa butter-like fat stitutes by means of lipase-catalyzed intersterification has at- . Acetone fractiona- tracted recent attention (6–10) owing to the availability of li- tion of the interesterified blends was performed using a modi- pases that catalyze the regioselective interchange of acyl fication of the procedures reported previously (4,5). Acetone groups at the 1- and 3-positions of the TAG structure, and (7.5 mL/g of substrate) was added to the fat mixture at the end such substitutes have been approved for food use (2). of the interesterification reaction. The warm acetone solution was quickly filtered through filter paper to remove the en- zyme, the filtrate was cooled to room temperature (≈22°C), *To whom correspondence should be addressed at USDA, ARS, ERRC, 600 East Mermaid Ln., Wyndmoor, PA 19038. and any precipitated solids were removed by filtration. The E-mail: [email protected] filtrate was then cooled to 4°C for 4 h, and the precipitated Copyright © 2003 by AOCS Press 1193 JAOCS, Vol. 80, no. 12 (2003) 1194 R.D. ABIGOR ET AL. TAG crystals were filtered at 4°C to give the cocoa butter-like The carrier gas was helium with linear velocity of 22.9 fat. The crystallized products were then subjected to GLC, cm s−1 at 80:1 split ratio. Elution of FAME was carried out RP-HPLC, HPLC-MS, and DSC analyses. with temperature programming from 140 to 155°C at 0.5°C (iii) Purification of cocoa butter-like fats. Silica gel 60 min−1, then 155 to 200°C at 20°C min−1. FAME were identi- (17.5 g, 40–63 µm particle size; Macherey-Nagel Polygosil fied by comparison with standards, mixture M-100, obtained 60-4063; Düren, Germany) was packed into a chromatogra- from Nu-Chek-Prep (Elysian, MN). phy column (4 cm o.d. × 120 cm) that contained a glass wool (v) RP-HPLC analysis. HPLC was carried out with a plug covered with sea sand. A mixture of hexane/ether (200 Hewlett-Packard model 1050 high-performance liquid chro- mL of 85:15, vol/vol) was passed through the column at a matograph equipped with an autosampler, quaternary pump flow rate of 2 mL/min under a slight nitrogen head pressure module, and a Varex (Burtonville, MD) ELSD II mass detec- (void volume of the column was approximately 90 mL). The tor. The detector output was routed to a Hewlett-Packard Vec- 4°C crystallized fat sample (1.27 g) dissolved in hexane/ether tra XM24/100 MHz computer. The TAG mixtures were sepa- (8 mL, 85:15) was then applied to the head of the column. Ex- rated by nonaqueous RP-HPLC on a Beckman/Altex (Rainin, ternal heat with a heating tape was used at the top of the col- Woburn, MA) Ultrasphere ODS (5 µm) column (250 × 4.6 umn to maintain dissolution of the solid TAG. The column mm). Separations were carried out with acetone (solvent A) was eluted with hexane/ether (85:15, 600 mL) at a flow rate and acetonitrile (solvent B) as eluents at a flow rate of 0.8 of about 2 mL/min, fractions were collected (20 × 15 mL and mL/min and the following solvent gradient profile: initial 10 × 30 mL), and the solvent was removed with a rotary evap- condition 70:30 (volume percent A/B), hold for 5 min, to orator. Each fraction was reconstituted with ether (1 mL) and 100% A over 15 min, hold for 8 min, return to original condi- analyzed on a silica TLC plate (10 × 20 cm, 250 µm). Plates tions over 5 min, then hold for 5 min (total run time was 33 were developed with a solvent system composed of hexane/ min). Estimation of total TAG equivalent carbon numbers ether/acetic acid (80:20:1, by vol). After air-drying, the plates (13) for separated peaks was made by comparison with stan- were sprayed with methanolic sulfuric acid (10%) and heated dard TAG mixture G-1 (Nu-Chek-Prep). Characterization of for visualization of lipid classes. Fractions containing only TAG molecular species (14) was carried out on a Hewlett- TAG were combined, and solvent was removed under a Packard Model 5989 LC-MS with 59987A electrospray LC- stream of nitrogen to give the purified fat product. The puri- MS interface operated in the atmospheric pressure chemical fied blends (B1–5) obtained with the various mole ratios of re- ionization mode with the following parameters: N2 drying actants used are listed in Table 1. gas, 350°C, 13 mL; N2 nebulizing gas, 350°C, 50 psi; quadra- (iv) GC analyses. The fatty acyl composition of the RBD- pole temperature 150°C, ionizing voltage 100 V; HPLC con- PO, HSO, and fat products (interesterified blends) was ditions used in LC-MS were as described earlier. determined by converting their TAG into FAME (13). The (vi) DSC analysis. Melting profiles for the TAG products FAME were analyzed by GLC on a Hewlett-Packard (Avon- were obtained with a PerkinElmer Pyris 1 differential scan- dale, PA) model 5895 chromatograph equipped with a split ning calorimeter (PerkinElmer Corporation, Norwalk, CT). capillary injector, an FID, and a Hewlett-Packard (model The samples (5–10 mg) were accurately weighed into alu- 3396) integrator.