Lipase-mediated interesterication of oils and fats for the preparation of bakery and confectionery fats

Bakery and confectionary fats were prepared by enzymatic interesterication of sal fat with palm stearin and palm mid fraction blends in various ratios. Slip melting point, free fatty acids, fatty acid composition, solid fat content and triglyceride composition were determined. Fatty acid composition revealed that the blends were rich in palmitic (13.9-58.5%), stearic (7.7-36.7%) and oleic (25.2-39.9%) with no trans fatty acids. Blends of sal:PSt (50:50), sal:PMF (50:50 and 25:75) showed high solid fat content at 20 and 25 °C with short melting range. After interesterication, plasticity of products increased, which were comparable with commercial bakery fat. Some of the blends alone showed short melting prole like cocoa butter. Interesterication produced signicant alteration in the triacylglycerol composition of the blends studied. Blends and the interesteried products prepared showed favorable characteristics with no transfats and hence could be used in the place of commercial bakery and confectionary fats.


Introduction
Most natural oil and fat present, have limited application in their unaltered forms, which is imposed by their particular composition in fatty acids and triacylglycerols. The fatty acids are not randomly distributed in triacylglycerols (TAGs) of natural fats (Martini et al., 2009). The production of some kinds of edible fats requires fat blends that are able to impart plasticity to products such as margarine and shortening. To achieve these properties, fat blends may be modi ed by chemical or enzymatic interesteri cation (EIE) which causes modi cations in the properties of natural fats. Lipase-catalyzed interesteri cation is one of the alternative technologies for replacing conventional hydrogenation process to reduce or eliminate trans-fatty acids (Hunter, 2004). Because of the increasing concern about the nutritional impact of trans-fatty acids on health, interesteri cation has become the main method for the preparation of plastic fat with low or zero trans isomers.
Interesteri cation (EIE), which offers the possibility of manipulating the distribution and composition of fatty acids of TAGs to confer more desirable physicochemical properties on fats, has been widely used as a fat modi cation process to provide plastic fats of desirable qualities (Norizzah et al., 2004;Chu et al., 2002). In the interesteri cation reaction, fatty acids remain unaltered, although they are redistributed in the triacylglycerol molecules.
Palm oil is readily available low cost fat and suitable raw material for the production of cocoa butter equivalent (CBE).
Preparation of CBE through enzyme catalyzed interesteri cation has attracted because lipases offer several advantages over other chemical catalysts such as, milder conditions, produces fewer by-products and regio-speci city (Willis et al., 1998). Other advantages are lower energy consumption and better product control, moreover solvent-free reaction has been considered as eco-friendly and economical. While chemical catalysts will randomize all of the fatty acids in triacylglycerol (TAG) mixture, 1, 3speci c lipase can incorporate fatty acids into the sn-1, 3-positions without changing the fatty acid residues in the sn-2position (Chang et al., 1990;Daniel et al., 2001;Zarringhalami et al., 2010). Vegetable oils such as mahua, kokum and mango fats, palm oil mid fraction, tea seed oil, and olive oil have been popularly used to prepare CBE by microbial lipases in batch stirred tank reactor (Wisdom et al., 1986). Pinyaphong and Phutrakul (2009) have modi ed palm oil structure to cocoa butter equivalent by Carica papaya lipase-catalyzed interesteri cation. However, procedures of lipase-catalyzed interesteri cation for CBE production have not been practically in industries because microbial lipase is relatively of high cost. Non-speci c lipase catalysed interesteri cation is similar to random chemical interesteri cation (Boleslaw et al., 2004). Random interesteri cation was used to prepare low or zero trans plastic fats from blend of liquid oils and hard fats (Martini et al., 2009;Norizzah et al., 2004;Mayamol et al., 2009;Reshma et al., 2008;Farmani et al., 2007;Khatoon, 2000).
Palm oil mid-fraction (liquid/solid state) characteristics are used in the production of confectionary for the replacement of fats such as cocoa butter and margarine. Lipase-catalysed interesteri cation has been carried out with high oleic sun ower oil (SFO) and fully hydrogenated soybean oil (Adhikari et al., 2009), palm stearin and rice bran oil blends (Reshma et al., 2008), fully hydrogenated soybean, rapeseed and SFO (Farmani et al., 2007), palm stearin-SFO mixtures (Lai et al., 1998), hydrogenated and solid fraction of tea seed oil (Zarringhalami et al., 2010).
Currently plastic fats are prepared by hydrogenation of vegetable oils and are characterized by high contents of trans fatty acids (20-50%) depending on the degree of hydrogenation and the nature of the oils. Trans fatty acids reportedly contribute to several health problems including thrombogenesis that leads to coronary heart disease (Willett et al., 1993). The interesteri cation provides higher quality plastic fats with satisfactory melting properties and suitable crystallization behavior with little sandy mouth feel.
Plastic fats having varying melting or plastic ranges are used as bakery shortenings, and margarines. Shortenings provide desirable textural properties by lubricating, weakening or shortening food components. Preparation of plastic shortenings from vegetable fats and oils by chemical, enzymatic interesteri cation and fractionation followed by blending are reported (Wang & Shahidi, 2011;Zahra & Alemzadeh, 2011;Khatoon & Reddy, 2005;Wai et al., 2007;Reddy & Jeyarani, 2001). Six binary formulations of medium-and long-chain triacylglycerols (MLCT fat) and palm stearin and four ternary formulations of MLCT fat, palm stearin and palm olein were investigated for shortening production (Ari n et al., 2011).
In the present study, preparation of bakery and confectionery fats were attempted using non-traditional and traditional fats/oils viz., sal fat and palm oil fractions. Different fractions of palm oil viz palm stearin (PSt) and palm mid fractions (PMF) were blended with sal fat. Sal fat, a non-traditional fat, is hard, brittle and closely resembles cocoa butter (CB). Palm stearin which is a by-product of palm oil industry and is the hard fraction obtained by palm oil fractionation, was taken to improve tolerance to high temperatures, for crystal morphology and also stability (Chu et al., 2002). Palm mid fraction was obtained by two stage crystallization. Sal fat and plam oil were considered for the experiments because palm oil is rich in POP triglyceride and sal oil with SOS triglyceride, which offers required melting pro le to the end products.

Materials
Crude palm oil was purchased from M/s Palm Tech. India Ltd. (Mysore, Karnataka, India) and Sal (Shorea robusta) fat was procured from M/s K.N. Oil Industries, Mahasamund, M.P., India. Crude sal fat and palm oil were re ned and bleached in laboratory as described by Hodgson (1996). Commercial bakery shortenings were procured from M/s Hindustan Lever Ltd., Mumbai, India. Immobilized 1, 3-speci c lipase, Lipozyme® RMIM from Rhizomucor miehei, was procured from Novozymes, Bangalore, India. Standards for the estimation of fatty acid methyl esters and triglycerides were procured from Sigma-Aldrich Co., St. Louis, MO, USA. Other solvents and reagents were of analytical grade.

Fractionation and blending
Crude palm oil was re ned, heated to about 50 °C, gradually cooled to 25 °C with stirring and this temperature was maintained for 4h. The partially crystallized mass was ltered to separate palmstearin fraction (PSt) and palm mid fraction (PMF). Fractionation yielded 35% Pst and 40% PMF was obtained by the removal of 8% PSt. Palm stearin and palm mid fraction were blended with re ned sal fat in various ratios viz., 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40 and 50:50. The melted blends were held at 100 ºC under vacuum with stirring for 10 min and homogenized prior interesteri cation. Promising blends were screened for speci c end use.

Interesteri cation
Lipozyme RMIM (10% by weight of oil) was used to carry out the interesteri cation reaction of puri ed oil blend (100 g) at 50 °C with continuous stirring on a magnetic stirrer. The reaction was carried out upto 8h, the products were drawn at 2, 4, 6 and 8h intervals either in presence or absence of water or in vacuum. The reaction products were ltered and the enzyme was recovered by washing with hexane. Free fatty acids which were generated during the reaction were neutralized using calculated amount of alkali and each product was ltered using lter aid and nally de-solventized under vacuum.
Physico-chemical analysis of blends and interesteri ed products Fatty acid composition The fatty acids methyl esters were prepared using methanolic KOH according to AOCS method Ce 2-66 (AOCS, 2002) and analyzed by gas liquid chromatograph (GC-15A, Simadzu Corporation, Kyoto, Japan) equipped with data processor (CR-4A, Simadzu Corporation, Kyoto, Japan) and ame ionization detector. The column used was stainless steel (3 m× 3.3 mm) coated with 15% DEGS (Diethylene Glycol Succinate) on Chromosorb W (60-80 mesh). Analysis was carried out under the following conditions; nitrogen ow: 40 mL/min, column temperature: 180 °C, injector temperature: 220 °C, detector temperature: 230 °C. Standard fatty acid methyl esters were used to identify the fatty acids in the blends.

Slip melting point (SMP)
Slip melting point of the samples was carried out by open capillary method Cc 4-25 according to standard procedure (AOCS, 2002). The sample was melted and a capillary tube with thin wall and 1 mm i.d was dipped to ll fat for 10 mm height.
The capillary was touched over a piece of ice to solidify the fat. The capillary was left at refrigerated temperature (10 °C) for 10 h and at 0 °C for 1 h. Two capillaries were attached gently to a thermometer using a rubber band and xed onto a Thiele tube.
The side arm of the ask was heated slowly (1 °C/min) and the temperature at which the fat melts, slips and raises was noted.
Triplicate measurements were made and the average value was reported.

Differential Scanning Calorimetry (DSC)
Melting characteristics of the samples were determined using a Mettler differential scanning calorimeter (Zurich, Switzerland, DSC-30). Indium was used to calibrate the heat ow of the instrument. All the samples were heated to 60 °C to ensure homogeneity and to destroy all crystal nuclei. Molten sample (15 mg) was accurately weighed into standard aluminum crucible and cover crimped in place. An empty aluminum crucible with pierced lid served as reference. The samples were stabilized by keeping at 0 °C for 90 min, 26 °C for 40 h and 0 °C for 90 min prior to introduction into DSC cell (IUPAC, 1987). Thermograms were recorded by heating the sample at the rate of 2 °C/ min from -10 to 60 °C. The percentage of liquid at various temperatures was recorded directly using data processor TC-10A and STARe program. The solid fat content (SFC) was calculated from the percentage of liquid.

Statistical data analysis
Experimental results were carried out in triplicates, and the results are shown as mean value and standard deviation.
Duncan's multiple range test (DMRT) was applied to differentiate the solid fat content and triglycerides species among the means of different samples at a probability (P) of ≤ 0.05 (Duncan, 1995).

Results And Discussion
Effect of enzymatic interesteri cation (EIE) on physico-chemical properties of sal blends with palm stearin and palm mid fraction Slip melting point (SMP) Blends containing high sal fat percentage (85 to 60%) showed increase in SMP and those of high palm stearin blends (60 and 80%) showed decrease in SMP after interesteri cation and also the effect depended on time of reaction. SMP decreased in the blends with equal proportion of sal and PSt and increased with the interesteri cation time. Sal:PMF blends followed the same trend as sal:PSt. It is known that SMP is only an indication of nal melting of the sample and does not show the entire melting pro les, which is required to assess the suitability of the sample for any end product.

Free fatty acid (FFA)
It was observed that there was a sudden increase in FFA during interesteri cation for 2h and thereafter increase was not signi cant (Fig. 1a). FFA formation was less in the products (Fig. 1b) prepared without added moisture (increased from 0 to 5% in 2 h) as compared to those with added moisture (increased from 0 to 16% in 2 h). FFA formation under vacuum was comparatively less than that with the solvent.

Fatty acid composition
All the blends of sal fat and PSt/PMF were rich in palmitic (13.9-58.5%), stearic (7.7-36.7%) and oleic acids (25.2-47.1%). Fatty acid composition varied depending on the ratio and yield percentage of fractions used for blending and all did not contain any trans fatty acids (Table 1).
Palm stearin is known to promote the formation of desirable solid fat having b ' crystal when it was interesteri ed with vegetable oils resulting in smooth textured products (Ming et al., 1999;Ghotra et al., 2002). The β′-crystal form is more stable in shortenings with higher palmitic acid contents (Jeyarani & Reddy, 2003). Due to high palmitic acid content these samples impart a desirable smooth consistency required for bakery shortenings. The fatty acid composition of the interesteri ed products is not shown as interesteri cation did not alter the fatty acid composition and only alters the distribution of fatty acid on glycerol backbone (Lida et al., 2007;Allen, 1996).

Solid fat content (SFC)
The SFC of fats is responsible for functional characteristics of plastic fats, including their general appearance, ease of packing, spreadability, oil exudation and organoleptic properties. SFC at 20 °C and 30 °C in particular are important in margarine manufacture, and must be as low as possible to prevent a sandy and coarse texture of the margarine (Osorioa et al., 2006). The plasticity or SFC varied depending on the proportion of palm stearin. Blends with higher proportions of palm stearin were harder than those with lower proportions. Blends containing sal and PSt showed increased plasticity with lower SFC at ambient temperature after interesteri cation (Table 2). Aquedo et al., (2008) reported that enzymatic interesteri cation (EIE) of anhydrous milk fat with linseed and rapeseed oil in the ratio 70/30 and 60/40 resulted in plastic fats with decreased SFC.
The blends of sal: PSt (75-85 to 15-25) and sal with PMF (50-75 sal) showed short melting range with high SFI at ambient temperature with no solids at body temperature like those of cocoa butter (CB) ( Table 2 & Fig. 2a & b). The blends with higher percentage of PSt showed higher SFC at and above 35 °C. Hence, these blends of PSt and PMF with sal alone could be used as cocoa butter extenders.
On interesteri cation, these blends of sal with PSt or PMF changed into long melting range with less SFC at 20-25 °C and extending solids at and above 40 °C (Table 2). Similar results were observed by De Martini et al., (2012) with blends of palm stearin, coconut and canola oil. The interesteri ed blends of sal:PSt (75:25/8h) and sal:PMF (50:50/6h) showed long melting range like plastic fats and are similar to those of commercial hydrogenated fat used for bakery products (Fig. 2a & b).

Triglyceride composition
The molecular triacylglycerol (TAG) species pro le is the key for understanding several physical properties of a given oil or fat. According to the laws of probability, the interesteri cation reaction results in complete fatty acid randomization among all triacylglycerols present (Rousseau & Marangoni, 2002).
The Interesteri cation produced signi cant alteration in the TAG composition of the blends. Palm stearin and sal blends (50:50) showed increase in trisaturated glycerides (GS 3 ) and decrease in monosaturated diunsaturated glycerides (GSU 2 ) and hence SFC at 35°C and above increased after interesteri cation. The GS 3 and GSU 2 triglycerides increased in 65:35 blends after EIE and resulted in increased SFI at 37°C and above (Table 3). Signi cant difference was observed in the slip melting point of Sal:PSt (75:25) after interesteri cation, where GS 3 increased, while disaturated monounsaturated glycerides (GS 2 U) decreased compared to those in the blend. Results showed that after interesteri cation for different time intervals tri-saturated glycerides were reduced and di-unsaturated increased in Sal:PSt (20:80) blend (Table 4). The observed physical properties like SMP and SFC are due to changes in TAG composition after interesteri cation.
In case of sal and PMF blends after 2h reaction, the product did not show any difference in GU 3 triglycerides compared to parent blend and GS 3, GSU 2 not showed any signi cant increase, while GS 2 U reduced (Table 5). Due to changes in triglyceride composition, SFC reduced at 20 -25 °C in all these blends.

Conclusion
Trans free bakery and confectionary fats were prepared by enzymatic interesteri cation of blends of sal with PSt and PMF.
Palm stearin and sal fat, a non-traditional fat, used in the present study are of low cost, at the same time they offer technical advantages and improved quality with the added health bene t. Blends prepared with sal and palm oil fractions (PSt and PMF) could be used as cocoa butter replacers even in tropical climatic conditions. Interesteri cation of sal blends with PSt/PMF produced products with desirable low SFC, long plasticity comparable with that of commercial butter and can also be used as margarine base.   Table 3 Triglyceride composition of sal and fractions of palm oil blends and interesterified products