13th ASEAN Food Conference, 9-11 September 2013, Singapore Meeting Future Food Demands: Security & Sustainability

Production of Cocoa Butter Substitute via Interesterification of Mango seed oil

Elham Momeny1,*, Leila Najafian1, Shahrooz Rahmati1, Nazaruddin Ramli1
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School of Chemical Science & Food Technology, Faculty of Science & Technology, University Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia * Corresponding author: elham_mn81@yahoo.com

Abstract In this study, production cocoa butter substitute through the enzymatic interesterification between Malaysian mango seed oil (MSO) and cocoa butter (CB) in three different ratios 60:40, 50:50 and 40:60 were carried out. The slip melting point, saponification value and iodine value of the 40:60 blend were 28.75C, 186.8 and 36.3 respectively. Analysis was shown that the 40:60 blend was close to cocoa butter, compared to the other enzymatic and non-enzymatic blends. The thermal behavior of the 40:60 blend showed that crystallization occurred between 18.73C and -52.55C, with a Hc of 89.74 J/g, and melting between -8.72C and 45.56C. The fusion maximum was 24.79C with fusion enthalpy 77.04 J/g. The 40:60 MSO: CB blend had a profile similar to that of CB. These results indicate that Malaysian mango seed oil has potential in synthesis lipid which has similar properties to be used in chocolate and confectionery products. Keywords Mango seed oil (MSO), Enzymatic interesterification, Thermal behavior, Cocoa butter (CB), Cocoa butter substitute (CBS)

1. Introduction
Mangos (Mangifera indica L.) are one of the fruits of high economic importance in Malaysia. Particularly, the Chacanan variety, which has been used as raw material for many canned fruit products. After utilizing mango by those factories, the seeds are discarded as the main waste and by-product of the mango juice industry [1]. The percentage of oil in the seed is about 8-12%. It is used as a component for CBE after fractionation. Mango kernel has a very big mango stone that makes the business to be more economical [16]. In recent years, the lipid composition of mango seed kernels has attracted scientists attention. One of the reasons is its unique physical and chemical characteristics. Extraction and fractionation of total lipid from Alphonso mango kernel have been carried out [18,19]. Mango kernels have high stearic acid content. Total lipid is 11.6% (of a dry kernel). This is made up of 96.1% natural and 3.9% polar lipids of which 2.9% is glycolipids while the phospholipid is about 1%. The natural lipids vary from 95.2% to 96.2%. Triglycerides constitutes the major fraction of natural lipid in mango kernel, It is made up of between 44-48% saturated fatty acid (majority stearic) and 52-56% unsaturated (majority oleic) [2,12]. The nutritional studies of mango seed kernel indicated that mango seed kernel fat is a promising and safe source of edible oil. The components of mango seed oil are commercially valuable in the vegetable oil and confectionery industries [3]. Cocoa butter is introduced as a unique simple fat obtained from pressing ground, roasted, decorticated cocoa beans [5]. In the liquid state, it has a bright yellow colour but yellowish white in solid form. It has a typical composition of 26% palmitic acid, 36% stearic acid, 33% oleic acid and 3% linoleic acid, although there are slight variations in the composition depending on the geographical source [9]. Cocoa butter is widely for the production of chocolate and other confectionery products, and to a limited extent, in the pharmaceutical industry as theobrorna oil [5]. Cocoa butter is particularly suitable for these purposes because of its low but sharp melting point, it is brittle and fractures readily, and it is not greasy when touched. It melts completely in the mouth [5]. All these properties are a reflection of its triacylglycerol composition comprising mainly of 1 -palmitoyl-2oleoyl-3-steamy1 glycerol (POS), 1, 3-distearoyl-2-oleoyi glycerol (SOS) and 1, 3-dipalmitoyl-2Oleoy1 glycerol (POP). Enzymatic interesterification has been employed by industry for the production of cocoa butter alternative and human milk fat substitutes (Quinlan &
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13th ASEAN Food Conference, 9-11 September 2013, Singapore Meeting Future Food Demands: Security & Sustainability

Moore 1993). Cocoa butter triacyglyceride mainly include 1,3-dipalmitoyl-2- oleoylglycerol (POP) (12.26%), 1-palmitoyl-2oleoyl-3-stearoyl glycerol (POS) (41.2%) and 1,3-distearoyl-2- oleoyl glycerol (SOS) (27.54%) [9]. With the use of 1, 3-specific lipases, it is possible to produce valuable cocoa butter substitute/equivalent from cheaper starting materials through interesterification [7,4, 8,6]. Since the price of fats has varied considerably over the years, but cocoa butter is one of the most expensive. The high and variable cost has stimulated an extensive search for fats that could be used as substitutes for or to replace cocoa butter in the industries. Attempts have been made to produce substitutes having same characteristics with cocoa butter using cheaper and available oil sources [6]. These are introduced variously as cocoa butter replacement fat, cocoa butter substitutes and cocoa butter equivalents. Cocoa butter substitutes are not chemically same as cocoa butter, but they are compatible with cocoa butter only within specified limits. In this research, mango seed oil was utilized in synthesizing cocoa butter substitute through enzymatic interesterification.

2. Materials and methods

2.1. Materials Fresh and mature chokanan mango collected from open market in Serdang Malaysia. Cocoa butter (CB) was obtained from KL kepong Sdn Bhd Port Klang, Malaysia. The Lipozyme TL IM (Immobilised 1, 3-specific lipase) was from Thermomyces lanuginose. (Novozymes A/S, Bagsvaerd, Denmark). 2.2. Methods 2.2.1. Preparation mango seed kernel All the fruit was peeled and removed pulp completely. The seeds were washed and air dried. Kernels were removed from seed manually, and chopped and dried [20]. Drying should continue until the moisture content below 10%. The dried kernels were ground in hammer mill with 100 mesh sieve into powdery form and kept in the closed dark glass bottle. They were stored at 4C until utilization. 2.2.2. Extraction oil from mango seed Extraction oil from mango seed was carried out by soxhlet extractor. First pack 100g of the shelled and ground seed meal into weighted thimble or cloth then introduced into extractor. Solvent was used is hexane. The temperature depends on boiling point of solvent in use, which was 140C for 6 to 7 hours [15]. 2.2.3. Purification of mango seed oil (MSO) Mango seed oil from mature fruit was purified using an adaptation of the Wesson method [10].

2.2.4. Interesterification reaction system Interesterification reaction was based on Macrae (1983) method. 2.2.5. Thermal profile
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13th ASEAN Food Conference, 9-11 September 2013, Singapore Meeting Future Food Demands: Security & Sustainability

Purified samples CB, (MSO: CB) 50:50 were fed into differential scan calorimeter (DSC, USA). Nitrogen gas was utilized at a flow of 20mL/min. Calibration instrument with indium (melting point, 156.6C; Hf, 28.45J/g). Samples were weighed around 4mg by thermobalence, in aluminium solid fat index (SFI) capsules (precision of 0.1 mg). Capsules were sealed hermetically. Before determination thermal profile, samples tempered by heating at 90C for 5 min after that cooled at 24C for 24h. Fusion profile by cooling -60 C and heating at 10 C/min to 90C registered. Crystallization profile by heating at 90C for 10 C/min and cooling at -60C registered. The fusion enthalpies required melt fat crystals, in addition the crystallization enthalpies required to solidify them, so enthalpies were calculated utilizing the area below the integration curve also the thermogram baselines. Range of temperature for fusion and crystallization became clear in the curve of the onset and offset temperature through phase changes [15]. 2.2.6. Solid fat content The solid fat content of samples was determined by using Bruker Minispec Mq 20 NMR Analyzer (Rheinstetten, Germany). This procedure was based on the IUPAC 2.150 method (1987). All the samples in NMR tube were melted at 80C and held for 30 min at 60 C. Samples were cooled at 0C for 90 min and after that held at 26.5C for 40h and then cooling for 90 min at 0C. All samples should be stabilized at (10, 20, 25, 30, 35 and 40 C) for 60 min before measuring liquid signal of every sample [14]. 2.2.7. Slip melting point This method is based on PORIM test no. p 4.2, (1995). 2.2.8. Iodine value The number of grams of iodine absorbed by 100g of the fat under the test condition is expressed as iodine value; this is according to PORIM test method no. p3.2, (1995). 2.2.9. Saponification value The number of the milligrams of potassium hydroxide required to soponify1g of sample under the condition specified is expressed as the saponification value (SV). This method is based on PORIM test no. p3.1, (1995).

3. Results and discussion

Determination slip melting point is necessary to determine, which fat blend is suitable to be CBS, Table 1 exhibits SMP of all blends with and without using enzymes. 40:60 blend has no significant different with CB after enzymatic reaction and other blends are significantly different from SMP of CB (P<0.05). Slip melting point of mango seed kernel (Mangifera Indica) was determined 35 C [11]; however, Abdalla et al (2007) reported that the melting point of Egyptian mango fat was 30.50 C. According to Table 1 the highest saponification value (SV) in all blends belongs to (0:100), which is pure CB, while amount of CB in blends increased, SV increased proportionally. SV of cocoa butter was not significantly different from SV of (40:60) blends with enzyme and without enzyme. Based on Abdalla et al (2007) results, saponification value of Egyptian mango seed oil was 192.16 (mg KOH/g Oil), which is in agreement with SV of MSO: CB mixture with portion 40:60 in this study. According to result shows in Table 1 IVs of all mixtures are significantly higher than CB
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13th ASEAN Food Conference, 9-11 September 2013, Singapore Meeting Future Food Demands: Security & Sustainability

so, cocoa butter with smallest unsaturated fatty acid had hardest fat. The earlier study by Moharram & Moustafa (1982) found that IV of mango seed kernel oil (Mangifera Indica) 49 (g I2/100g Oil) while the recent studies [3] found that 53 (g I2/100g Oil) which is in agreement with result of current study.
Table 1. Physicochemical analysis of interesterified blends of MSO:CB Blends (MSO:CB) SMP (C) IV (gI2/100Oil) SV(mgKOH/gOil) 100:0 60:40 50:50 40:60 0:100 18.13 0.32e 22.5 0.5d 27.43 0.4c 28.75 0.25b 30.23 0.25a 50.7 0.29a 42.43 0.4b 39.54 0.13c 36.3 0.4d 34.13 0.32e 138.52 0.5e 160.06 0.2d 178.4 0.45c 186.8 0.6b 188.75 0.23a

According to plot SFC versus temperature in figures 1 melting behavior of the fat mixtures between 15-40C shows. {50:50} and {60:40} blends had melting behavior (at 30C) closed to cocoa butter. According to Solis-Fuentes et al (2004) mango almond fat was softer than cocoa butter at lower temperature and at 35 C shown sharp melting behavior which is close to MSO:CB mixture behavior in this research. Figure 1 present solid fat content diagram for interesterified mixtures of MSO: CB.

Figure 1. Solid fat content * (SFC ;%) of fat mixtures, Enzymatic reaction Note: *each mixture line represented the average of triplicates

The (MSO:CB) blend with {40:60} ratio had two maxima fusion point, first low fusion point with maximum temperature 13.78 C and the second with maximum temperature at 30.15C. Hf for this blend was 77.04J/g. The cooling process for (MSO: CB) blend stated at 18.73C and ended at -52.55C. Crystallization maxima were at 16.23, Hc for solidification was 89.74J/g which was shown in Figure 2.
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13th ASEAN Food Conference, 9-11 September 2013, Singapore Meeting Future Food Demands: Security & Sustainability

Figure 2. Crystallization and fusion curves for (MSO: CB) {40:60} which refer to table 2 for (1, 2, 3, 4 and 5) transition temperatures. Table 2. Transition point temperatures and crystallization and fusion enthalpies for (MSO:CB) and CB Samples H(J/g) 1 Crystallization MSO: CB{40:60} CB 89.74 84.86 18.73 16.31 16.23 10.61 -30.54 -21.22 -40.99 -32.17 -52.55 n.d. 2 Transition temperature (C) 3 4 5

Fusion MSO:CB{40:50} CB

77.04 73.35

-8.72 -10.02

13.78 12.60

24. 79 22.03

30.15 30.51

45.56 46.47

4. Conclusion
According to this research MSO:CB 40:60 blend have enough potential to be used as cocoa butter substitute in production chocolate and confectionery products

5. References
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13th ASEAN Food Conference, 9-11 September 2013, Singapore Meeting Future Food Demands: Security & Sustainability

[4] S. Bloomer, P. Adlercreutz, B. Mattiasson. Triglyceride Inter-esterification by lipases. I. Cocoa butter equivalents from fractions of palm oil. J Am Oil Chem Soc, 67 (1990) 519-524. [5] M. Gordon. Fats and fatty foods. Edited by M. D. R. a. K. R.C. Vol. 288-327, Food Industries Manual. London: Blackie Academic and Professional, 1993. [6] K.J. Liu, H.M. Chen, R.C. Chang, J.F. Shaw. Synthesis of cocoa butter equivalent by lipase-catalysed inter-esterification in supercritical carbon dioxide. J Am Oil Chem Soc, 74 (1997) 1477-1482. [7] A.R. Macrae.. Lipase catalysed inter-esterification of oil and fats. J Am Oil Chem Soc, 60 (1983) 291 - 294. [8] Mojovic, L., Siler-Marinkovic, S., Kukic, G., Bugarski, B & Vunjak Novakovic,G. Rhizopus arrhizus lipase-catalysed inter-esterificatioin of the mid fraction of palm oil to cocoa butter equivalent. ENZYME MICROB TECH, 15 (1993) 438-443. [9] J.Y. Owusu-Ansah. Enzymes in lipid technology and cocoa butter substitute. In Technological advance in improved and Alternative sources of lipids, edited by B. S. K. Kamel, Y. London: Blackie Academic and Professional, 1994. [10] Mehlenbacher, V.C. Analysis de grases y aceites. Ediciones Urmo, Bilbao,1970. [11] Y.G. Moharram, A.M. .Moustafa. Utilization of mango seed kernel (Mangifera Indica) as a source of oil. Food Che, 8 (1982) 269-276. [12] E. M. Mohamed, A.Y. Girgis.Utilization of mango seed kernels for improving stability of some oil and biscuit production. J. Agr. Sci. Tech, 30 (2005) 4625-4636. [13] PORIM. 1995. Palm Oil Research Institute of Malaysia (PORIM) test methods. Kuala Lumpur. [14] S. Soekopitojo, P. Hariyadi, T.R. Muchtadi, N. Andarwulan. Enzymatic interesterification of palm oil mid fraction blend for production of cocoa butter equivalent. Asian jouranal of food and agro-industry 2 (2009) 807-816. [15] J.A. Solis-Fuentes. M.C. Duran-de-Bazua. Mango seed uses: thermal behavior of mango seed almond fat and its mixtures with cocoa butter. Bioresour Technol, 92 (2004) 71-78. [16] Soon, Wong. Chocolate speciality fats the cocoa business. In getting to know your fats chemis, 2002. [17] P. Quinlan, S. Moore. Modification of triglycendes by Lipases, Process technology and its application to the production of nutritionally improved fats,1993. [18] J. Hemavathy, J.V. Prabhakar, D.P. Sen,. Drying and storage behaviour mango (mangoifera indica) and coposition of kernel fat. ASEAN Food J, 4 (1988) 59-63. [19] J. Hemavathy, J. V. Prabhakar, D. P. Sen. Composition of polar lipids of alphonoso mango (mangoferia indica) kernel. Food Sci, 52 (1987) 833-834. [20] M.A. Augustin, E.T. Ling.Composition of mango seed kernel. Pertanika, 10 (1987) 53-59.
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13th ASEAN Food Conference, 9-11 September 2013, Singapore Meeting Future Food Demands: Security & Sustainability