Blending of Mango Kernel Fat Stearine with Palm Oil Mid-fraction to Obtain Cocoa Butter Equivalent

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1 American Journal of Food Science and Nutrition Research 2017; 4(2): ISSN: X (Print); ISSN: (Online) Blending of Mango Kernel Fat Stearine with Palm Oil Mid-fraction to Obtain Cocoa Butter Equivalent Warda Mwinyi Pembe 1, 2, Abuubakar Hassan Ramadhan 1, Khamis Ali Omar 1, 2, Jun Jin 1, Jianhua Huang 1, Zheng Liyou 1, Qingzhe Jin 1, * 1 State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, School of Food Science and Technology, Jiangnan University, Wuxi, PR China 2 Ministry of Health, Zanzibar Food and Drugs Board, Zanzibar, Tanzania address warda_mwinyi@yahoo.com (W. M. Pembe), abuur@yahoo.com (A. H. Ramadhan), jqzwuxi@163.com (Qingzhe Jin) * Corresponding author To cite this article Warda Mwinyi Pembe, Abuubakar Hassan Ramadhan, Khamis Ali Omar, Jun Jin, Jianhua Huang, Zheng liyou, Qingzhe Jin. Blending of Mango Kernel Fat Stearine with Palm Oil Mid-fraction to Obtain Cocoa Butter Equivalent. American Journal of Food Science and Nutrition Research. Vol. 4, No. 2, 2017, pp Received: January 11, 2017; Accepted: February 4, 2017; Published: March 29, 2017 Abstract The physicochemical characteristics of mango kernel fat (MKF) and its fractionated fraction and the binary mixtures of the mango fat stearine (MFS) with palm oil mid fraction (PMF) were compared with cocoa butter (CB) for production of cocoa butter equivalent (CBE). Fat blends were prepared in five different weight ratios of MFS/PMF from 65/35 to 85/15 in five increment. All fat samples were evaluated for their thermal profile, solid fat content and their iodine values and slip melting points. All fats contain higher slip melting point ranging from 31.4 to 37.1 C then cocoa butter CB, in which the fat blends have higher then PMF but lower then MFS with their iodine values ranging from 34.0 to 35.4 mg/100g. Gas chromatographic analysis show that the major triacylglycerol of the blends are SOS (39.4 to 56.5%), POP (10.75 to 28.9%), POS (13.9 to 14.2%), which was similar to that of CB although in different amount. Also the major fatty acid compositions of the raw MKF and MFS are similar to that of CB those are stearic acid, oleic acid and palmitic acid. However blend 75/25 contains similar content of POP but high in SOS and lowers in POS then CB and blend 65/35 show melting offset temperature similar to that of CB (28.8 C). Indeed their SFC profiles were also comparable to that of CB. Keywords Cocoa Butter, Cocoa Butter Equivalent, Mango Fat Stearine, Solid Fat Content, Thermal Profile 1. Introduction Cocoa butter (CB) is the essential constituent in chocolate production, which in it absence cannot be a chocolate. It is extracted from cocoa bean, the seed of theobroma cocoa tree. Worldwide demands of CB have been increased and the price is high and can fluctuate in time, due to limited supply and crop yield [1]. The main fatty acids compositions of CB are stearic acid 33.6%, oleic acid 37.0%, palmitic acid 24.4%, 3.4%, lenoleic acid and others 1.6% [1]. These fatty acid build the three main triacylglycerols: glycerol-1,3- dipalmitate-2-oleate (POP; 15-19% ), glycerol-1-palmitate-2- oleate-3-stearate (POS 36-41% ) and glycerol-1,3-distearate- 2-oleate (SOS 25-31%) where stearic and palmitic are located at Sn- 1,3 position and oleic at Sn-2 position on triacylglycerol backbone that gives CB unique and desirable properties [2], [3]. The monounsaturated species and its polymorphic behavior make CB hard and brittle at room temperature while melting completely in the mouth [4]. The rapid increase of price and demands of CB force the researchers and industries to think on alternative that are cocoa butter alternative fats, which include, those exotic fats as mentioned by European Union s of Chocolate Regulations [5]. Among those fats are palm oil mid fraction, mango kernel fat, kokum fat, illipe butter, and others. Cocoa butter equivalents or extenders (CBEs) are vegetable fats containing the same fatty acids and symmetrical mono-unsaturated

2 72 Warda Mwinyi Pembe et al.: Blending of Mango Kernel Fat Stearine with Palm Oil Mid-fraction to Obtain Cocoa Butter Equivalent triacylglycerol as CB [5], [6]. They are fully compatible with CB and can be mixed with CB in any ratio in the chocolate formulations. CBE have been used to replace CB for many years using different techniques as observed from literature for example: refined bleached deodorized palm kernel oil or palm stearin for cocoa butter alternative [1], blending of mango kernel fat with palm oil mid fraction [7], palm kernel oil with milk fat [8], tea seed oil with methyl palmitate and methyl stearate [4] Mango (Mangifera indica, Linn) is the one among tropical exotic fruit of the family Anacardiaceae in the order of Sapindales. The fruit has a large, central stone, flattened, with a woody cover containing the kernel with a single or two embryos [9]. The kernel initially was regarded as waste by consumer and mango deal companies but currently can be used in production of several food products like the kernel powder can be mixed with all purpose flour for prepare biscuit and breads [10]. The seed, depending upon the varieties, can constitute 3-25% of the total mass of the fruit, and the kernel occupies 54-85% of the seed; it has moisture content of 33-86%, and the solid dried matter has proteins ( %), crude fiber %, ash content %, total carbohydrates of 33-76%, around % of phenolic compounds, and % crude fat [9]. Also the fat extracted from mango seeds, that is mango kernel fat (MKF) is the source of fat, which is hard at room temperature and has been6 determined to be suitable ingredients in confectionery and food industries with nutritional value and health benefit [11]. The study reported by [12], [13] using differential scanning calorimetry and nuclear magnetic resonance, they concluded that crystallization, melting profile and solid fat content of MKF is similar to that of CB. The solid fat content is physical parameter that used to indicate the hardness of the fat, fat with low solid fat content when used in confectionary products can results in soft products [14]. MKF have distinctive triacylglycerol composition dominated by SOS and SOO makes it easy to separate highmelting stearin fraction from the low melting fraction by fractionation and the resultant stearin fraction is further used in the mixture of CBE. Fractionation is the technique used in fat modifications especially CBE either its own or by mixing with other oil. This is the suitable technique to reduce amount of selected triacylglycerols and maintain other TAG in required amount necessary for blending with other vegetable fat or oil for CBE production. MKF fraction is a fraction of mango kernel fat obtained after acetone fractionation under appropriate conditions at specific time, which results into two fractions each with different physical and chemical properties (solid stearine and liquid olein). The mango fat stearine (MFS) also obtained after second fractionation of the fist solid stearine, it has melting point around C as reported by [15]. The CBE can be obtained by blending palm oil mid fraction (PMF) with MFS in several proportions and this is the crucial gat way for fat optimization. PMF is a fraction of palm oil that is obtained through refractionation, either from palm olein or palm stearin, and has melting point between 9.8 and 32.8 C [1]. It contain high content of symmetrical monounsaturated POP about 51.8%, PMF shows a steep solid fat content versus temperature curve, which is similar to that of CB and therefore is suitable in CBE production [1]. The objective of this study was to evaluate the physicochemical properties of the mixture of double fractionated mango kernel fat (MFS) with palm oil mid fraction in terms of their difference in triacylglycerols (TAGs), solid fat content (SFC), iodine values (IV), slip melting point (SMP) and thermal and crystallization profiles to produce high quality and heat resistance CBE. 2. Materials and Methods The dry mango seeds were obtained from local mango juice processing factories in Guangdong Province, South China. The kernels were removed manually and stored in the polyethylene bags in a desiccator prior to extraction. Commercial CB was purchased from Jinsihou group (Shanghai, China). PMF was imported from IOI palm oil production group in Malaysia. Standard of forty fatty acid methyl esters were purchased from Sigma-Aldrich Chemical Co. Ltd. (Shanghai, China). Triacylglycerol standards consisting of POP, POS and SOS (P, palmitic acid, S, stearic acid and O, oleic acid) were obtained from Larodan Fine Chemical AB (Malmö, Sweden). Other reagents and solvents were provided by Sinopharm Chemical Reagent (Shanghai, China) 2.1. Fat Extraction and Purification The crude fat was extracted from dried mango kernel powder by soxhlet technique using hexane in 1:5 (w/v) ratio at 60 C for 6 h, and refined as previously described by [15]. In order to attain maximum extraction, the process was duplicated with half quantity of solvent and all the extracts were pooled in the same round bottomed flask each time after evaporation of some solvent under vacuum by rotary evaporator at 45 C Fractionation Double fractionation of MKF was done by acetone in 1:5 (w/v) ratio, the first solid fraction was obtained at 13 C for 120 min of crystallization period. After filtration and separation with liquid under vacuum, and the first solid was then treated with acetone at same ratio and kept at 15 C for 90 min. The only solid fraction mango fat stearine (MFS) was used in further analysis CBE Preparation After purification and fractionation the clear yellow mango fats were obtained and prepared for blending, the mango fat stearine (MFS) was blended with PMF into five proportions of MFS/PMF: 65/35, 70/30, 75/25, 80/20, and 85/15 each in (w/w). All blends were analyzed simultaneously with, pure PMF, MKF, MFS and CB.

3 American Journal of Food Science and Nutrition Research 2017; 4(2): Fatty Acid Compositions of Raw Fats Fatty acid compositions of raw fats were determined using gas chromatographic system (7820A, Agilent, USA) after derivatizing the fatty acid into fatty acid methyl esters (FAMEs). Sample was prepared followed the method done by [16]. Separation was carried out on Trace TR-FAME capillary column (0.25 µm, 60 m Å ~ 0.25 mm, Thermo Fisher, USA) under the following conditions: 1 µl aliquot was injected into the column with a split ratio of 50:1. The injector and detector temperature were set at 250 C. nitrogen was used as carrier gas and set at 1 ml/min. the initial temperature was 120 C and hold for 3 min and then to 176 C at 8 C/min for 28 min, and then to 215 C at 3 C/min for 20 min. The fatty acid contents were given in terms of percentage area Triacylglycerol Determination Triacylglycerol compositions were determined by using gas chromatographic system (7890A, Agilent, USA) as explained by [14]. The GC was equipped with hydrogen flam ionization detector (FID). Nitrogen was used as the carrier gas at gas flow rate of 1mL/min and split ratio of 1:00. The enjector and detector temperatures were 360 C and 375 C respectively, the oven temperature was 340 C and maintained for 32 min. The content of each triacylglycerol was reported in terms of the relative proportion Slip Melting Point (SMP) and Iodine Values IV SMP was analyzed using method adopted from [7] with minor modifications. Briefly, three capillary tubes were filled with sample up to10 mm high, then left to solidify and refrigerated at 10 ± 1 C for 16 h. The capillary tubes were attached to a thermometer and suspended in a beaker containing distilled water. The water was stirred and the heat was applied, the temperature of the water was recorded when the fat column in each tube rises. Apart from that, Iodine values were analyzed using Wij s method as described by [17] Solid Fat Content SFC SFC was determined by pulse nuclear magnetic resonance pnmr (AM4000, Oxford, UK) following method adopted by [13]. Approximately 4 cm high of melted sample were filled in NMR tubes and held at 80 C for 60 min to erase crystal memory. There after, the SFC was measured in 5 C increments from 0 C holding for 90 min to 40 C following 30 min after each setting temperature Crystallization and Melting Characteristics Melting and crystallization profiles were obtained with a differential scanning calorimeter (DSC Q200, TA, USA) as adopted from [18] with minor modifications. Briefly, samples (8.0 ± 0.5 mg) were rapidly heated to 80 C and were held for 3 min to eliminate their thermal memory. The crystallization profiles were generated by cooling the samples from 80 to - 40 C at 10 C/min and holding for 5min, then the melting profiles were obtained by heating the samples to 80 C at 5 C/min. 3. Statistical Analysis The data were obtained in duplicate or triplicate and reported as mean and standard deviations. Analysis was carried out by SPSS program (version 19.0) at p < Results and Discussion Fatty acid composition of raw fats samples that are CB, MKF, MFS and PMF are presented in Table 1. CB used here comprised mainly stearic acid (S, 37.11%), oleic acid (O, 31.45%), and palmitic acid (P, 27.62%), which make up to 96.20% of total fatty acid. Similar to the cocoa butter reported by [1], [19]. Similar composition was observed in MKF and MFS; they make up to 91.74% and 95.26% respectively of the total fatty acid although MFS had oleic and lenoleic acid close with that of CB. MKF have high stearic acid compared with CB (S, 44.51%) and after fractionation the MFS contain (51.94%) this indicated MFS contained more saturated fatty acid compared to all fats and hence it was highly solid at ambient temperature. Oleic acid of MKF (37.01%) was slightly higher then CB and MFS (34.07%) had no significantly different with CB. Both MKF and MFS have smaller composition of palmitic acid about (10.22 and 9.25%) compared to CB. PMF had high content of palmitic acid (P 64.20%) followed by oleic acid (26.09%) and small quantity of stearic acid (5.54%). Thus PMF was useful in blending with high stearic acid fats to form appreciable cocoa butter equivalents. The PMF reported here was comparable to that reported by [16]. Table 1. Fatty acid compositions of mango kernel fat (MKF), mango fat stearine (MFS), cocoa butter (CB) and palm oil mid fraction (PMF) a. Fatty acid composition (Area %) Fatty acid MKF CB MFS PMF Myristic (C14) ND 0.18±0.00b ND 1.25±0.01a Palmitic (C16) 10.22±2.99c 27.62±0.05b 9.25±0.42c 64.20±0.26a Palmitoleic (C16:1) 0.17±0.01b 0.29±0.01a 0.21±0.00a 0.14±0.00b Stearic (C18) 44.51±1.83b 37.11±0.54c 51.94±0.74a 5.54±0.10d Oleic (C18:1) 37.01±2.71a 31.45±0.45b 34.07±0.22ab 26.09±0.08c Lonoleic (C18:2) 4.72±1.02a 2.03±0.08b 2.14±0.07b 0.22±0.01c Lenolenic (C18:3) 0.32±0.04b 0.10±0.01c 0.19±0.01c 2.27±0.06a Arachidic (C20) 2.18±1.09a 0.64±0.01c 1.54±0.02b 0.25±0.00d Behenic (C22) 0.40±0.12a 0.12±0.01c 0.32±0.01b 0.06±0.01d Lignoceric (C24) 0.36±0.03a 0.00± ±0.01a 0.00±0.00 a Mean ± SD with different letters in the same raw differed by at least 0.05 significant interval Triacylglycerol Composition TAG TAG compositions of MKF, MFS, PMF, CB and all blends are given in Table 2. Table 2 shows the main TAGs in CB are

4 74 Warda Mwinyi Pembe et al.: Blending of Mango Kernel Fat Stearine with Palm Oil Mid-fraction to Obtain Cocoa Butter Equivalent SOS (27.83%), POS (42.08%) and POP (17.69), which constituted (87.60%) of total triacylglycerol. The triacylglycerol compositions of MKF are similar to that of CB as obtained from literature. The MKF used here seem to contained (51.09%) amount of SOS, but after fractionation the content of SOS for MFS increased to (64.98%). Both MKF and MFS have higher amount of SOS compared with that of CB, which may be useful in tropical countries. The amount of POP and POS of both MKF and MFS were approximately equal to (1.70%) and (16.00%) respectively but were lower compared to that of CB (17.70% POP and 42.08% POS), similar CB composition was reported by [16]. The level of di-unsaturated TAGs was seemed to be reduced in MFS, example POO become approximately similar to that of CB and SOO become two times less than before fraction about (15.05 to 6.50%) this was because most of the olein move into liquid fraction. PMF had POP about 46.95% followed by POO (15.50%), POS (8.65%), PLP (8.40%), OOO (2.00%), MOP (2.00%), SOO (1.55%) and SOS (0.95%). This PMF after blending with MFS results into the new fats with different characteristics than corresponding raw fats. Triacylglycerol compositions of all fat blends were different among blends, the most abundant TAG are also POP, POS, SOS similar composition with CB. The trends were due to increase or decreasing the content of MFS or PMF. The content of SOS varies from (39.40 to 56.50%) as the amount of MFS increase and SOS increase proportionally while as increase amount of PMF the amount of POP rise from (10.75 to 28.90%). There was no big different in POS composition among the blends all lies from (13.90 to 14.20%). Blend 75/25 had approximately similar POP content with CB, while none of the blend exhibited same SOS and POS to that of CB. Table 2. Triacylglycerol compositions, iodine values (IV), slip melting point (SMP) for mango kernel fat (MKF), mango fat stearine (MFS), cocoa butter (CB), palm oil mid fraction (PMF) and different MFS/PMF blends. TAG 85/15 80/20 75/25 70/30 65/35 MKF MFS PMF CB MLP 0.05± ± ± ± ±0.07 ND ND 0.40±0.00 ND SOS 56.50± ± ± ± ± ± ± ± ±0.07 POP 10.75± ± ± ± ± ± ± ± ±0.27 SLO+PLS 0.15± ± ± ± ± ±0.32 ND 2.50± ±0.04 POS 14.20± ± ± ± ± ± ± ± ±0.02 PLO 0.10± ± ± ± ± ±0.24 ND 6.05± ±0.13 PLP 1.20± ± ± ± ± ± ± ± ±0.37 MOP 0.25± ± ± ± ±0.07 ND ND 2.00±0.00 ND OOO 2.60± ± ± ± ± ± ± ± ±0.13 POO 1.25± ± ± ± ± ± ± ± ±0.45 PPP 0.30± ± ± ± ±0.00 ND ND 1.80±0.28 ND SOO 5.65± ± ± ± ± ± ± ± ±0.21 PPS ND 0.10± ± ± ±0.00 ND ND 1.80±0.28 ND SOA 0.12± ± ± ± ± ± ±0.0 ND 1.08±0.03 PLL ND ND ND ND ND 0.03±0.04 ND 1.20± ±0.08 SMP ( C) 35.40± ± ± ± ± ± ± ± ±0.99 IV (mg/100g) 35.40± ± ± ± ± ± ± ± ±0.40 O oleic acid, S stearic acid, P palmitic acid, L linoleic acid, and M myristic acid. ND, not detected 4.2. Physicochemical Characteristics The iodine values (IV) and slips melting points (SMP) for the raw fats samples and the blends are shown in Table 2. The IV was a better indicator not only on hardness but also for the degree of unsaturation of the fat. The higher the IV in fat, the higher number of double bonds and hence the lower slip melting points. CB used here had IV of 34.8 mg/100g, this value was in between PMF (32.40 mg/100g) and MFS (36.00 mg/100g) while the MKF contain (36.90 mg/100g) therefore MKF had high amount of unsaturation compared to all fats. Indeed the high content of SOO and OOO on MKF as shown from Table 2 may the reason of this IV. Among the blends, the IV was increased from 65/35 (34.40 mg/100g) to 85/15 (35.40 mg/100g), although the trends were not liner. The IV of blends from 65/35 to 80/20 was approximately similar to that of CB Slip Melting Point SMP This is the temperature at which fat rises in an open capillary tube upon controlled heating. The SMP of CB (27.20 C) was smaller compared to those raw fats and the higher SMP was observed on MFS (37.10 C). Among the blends, all had higher SMP compared to CB and lower then that of MFS, Also the blend 70/30 contain the SMP closer with that of PMF. The SMP of the blends indicated that, the fat are useful in hot climatic regions around the world.

5 American Journal of Food Science and Nutrition Research 2017; 4(2): Crystallization and Melting Properties The melting and crystallization profiles are shown in Table 3 and Figure 1 and 2, CB display typically unique melting profile compare to other fats it was ranging from C to C and had lowest offset melting temperature compared to all fats (Fig not shown). Its melting offset temperature was not significantly different from that obtained in Table 2, this temperature was smaller compared to that reported by [16]. PMF had wide melting range (11.93 to C), indicating the presence of high levels of high and low melting triacylglycerol. Although its onset melting temperature was small compared to all fats, its offset temperature was comparable to that obtained in Table 2. Before fractionation the fat MKF had a wide melting range around (15.29 to C) due to the same reason explained above, its offset melting temperature was exact similar to that reported in Table 2. While after fractionation MFS, the sharp and narrow peak was observed within the range (24.09 to C). The higher temperature was observed due to the increase in amount of high melting TAG (SOS) and sharpness due to the reduction of tri-olein (OOO) and POO in the MFS as shown in Figure 1. Also PMF and CB possess significantly high enthalpy necessary to convert the solid into liquid compared with MKF and MFS. Interestingly the offset melting temperature of all fats were not exceeding C (body temperature), this means all fats were able to melt completely in the mouth. From the fat blends, as amount of PMF increases from (15% to 35%) while MFS decreased, the profile are become brooder and show variation in onset and offset melting temperatures. The offset melting temperature seemed to decrease as PMF amount increased from to C, the blend 70/30 and 65/35 had offset melting temperature close to that of CB. Figure 1. DSC melting thermograms of MKF, MFS, PMF and fat blends with different ratios of MFS/PMF. On the other hand, the crystallization of all raw fats was slightly different, MFS had lowest crystallization offset temperature ( C) this was due to presence of high content of disaturated triacylglycerol (SOS) as shown from Table 2, followed by PMF which was complete crystallized around C and MKF had C, the CB had the highest one C. MKF, CB and PMF showed wide crystallization range due to high content of high and low melting triacylglycerol compared to MFS (17.74 C to C). The blends followed the trends as amount of PMF increased while MFS decreased the onset and offset temperatures were decreased proportionally as shown in figure 2. In this case blend 85/55 have same crystallization onset temperature as that of CB.

6 76 Warda Mwinyi Pembe et al.: Blending of Mango Kernel Fat Stearine with Palm Oil Mid-fraction to Obtain Cocoa Butter Equivalent Figure 2. DSC crystallization thermograms of MKF, MFS, PMF and fat blends with different ratios of MFS/PMF. Table 3. Melting and crystallization profile for raw fats and blends. Melting Crystallization Sample Onset Offset Enthalpy Onset Offset Enthalpy CB MFS MKF PMF / / / / / Solid Fat Content (SFC) The profiles of SFC against temperature of raw CB, MKF, MFS and PMF and the all blends as measured by pnmr are shown in Figure 3. SFC gives the information about hardness and melting characteristic of the fat. All SFC for the fat samples were observed to decrease as temperature increase. MKF profile was differing from that of MFS, the lower SFC was observed to raw MKF, while in MFS the some amount of oleic acid was reduced and result to have high solid fat content. Also MFS show hardness from 0 to 35 C about (91 to 65%) and melting completely at 40 C while MKF was solid from 0 to 20 C, semisolid around 30 C and completely melt at 35 C this may be due to the nature of polymorphic behavior are changed, although this implies that the fats are melting completely in mouth (37 C). PMF display different SFC profile compared to MKF and MKS, this may be due to the presence of high content of high melting triacylglycerol compared to mango fat. Initially PMF exhibited high SFC compared to all fats, means it was strongly solid at bellow 25 C and become semisolid at 30 C and dramatically decrease from 30 to 35 C and melted completely above 35 C. Among blends there were no significantly deference in their SFC but lower than corresponding PMF and MFS fats and higher than MKF. This show that fractionation had improved the solid fat content of the fats. They exhibited more than 45% SFC at 25 C, and semisolid at 30 C while melted completely above 35 C. Therefore the blends show plastic properties and can be a good source of CBE in hot climatic regions.

7 American Journal of Food Science and Nutrition Research 2017; 4(2): Figure 3. Solid fat content of MKF, MFS, PMF and fat blends with different ratios of MFS/PMF measured at 0-40 C. 5. Conclusion Fractionated mango kernel fat obtained from Chinese mango varieties could be of interest to produce heat resistance edible fat. Compared with commercial CB, MFS had similar triacylglycerol composition with high content of SOS that makes the fat hard and attain high melting point. The modifications through blending prove the fats can be used as cocoa butter equivalent or improver. Melting and crystallization properties of the blends 70/30 and 65/35 are similar to CB although blend 85/15 also shows similar offset crystallization temperature. Similar to CB all fat blends showed the same solid fat profile, their solid fat content decrease as temperature increase, indeed blends 70/30 and 65/35 melt at same temperature as CB therefore they are recommended to be used as CBE in wide range of climatic conditions. References [1] Biswas N, Cheow YL, Tan CP, F. Siow L. Blending of Palm Mid-Fraction, Refined Bleached Deodorized Palm Kernel Oil or Palm Stearin for Cocoa Butter Alternative. J Am Oil Chem Soc [2] Soekopitojo S, Hariyadi P, Muchtadi TR, Andarwulan N. Enzymatic interesterification of palm oil midfraction blends for the production of cocoa butter equivalents. Asian Journal of Food and Agro-industry 2009; 2: [3] Kim S, Kim I-H, Akoh CC, Kim BH. Enzymatic Production of Cocoa Butter Equivalents High in 1Palmitoyl-2-oleoyl-3-stearin in Continuous Packed Bed Reactors. Journal of the American Oil Chemists' Society 2014; 91: [4] Wang H-X, Wu H, Ho C-T, Weng X-C. Cocoa butter equivalent from enzymatic interesterification of tea seed oil and fatty acid methyl esters. Food Chemistry 2006; 97: [5] Goodacre R, Anklam E. Fourier transform infrared spectroscopy and chemometrics as a tool for the rapid detection of other vegetable fats mixed in cocoa butter. Journal of the American Oil Chemists Society 2001; 78: Acknowledgment The research was financially supported by the Natural Science Foundation of Jiangsu Province (Grants No: BK ). Program of Science and Technology Department of Jiangsu Province (Grants No: BY ) and Graduate Research and Innovation Projects in Jiangsu Province.

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