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Extraction of cocoa butter alternative from kokum (Garcinia indica) kernel by three phase partitioning
Journal of Food Engineering 117 (2013) 464–466
Contents lists available at SciVerse ScienceDirect
Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Extraction of cocoa butter alternative from kokum (Garcinia indica) kernel by three phase partitioning Ganesh S. Vidhate, Rekha S. Singhal ⇑ Food Engineering and Technology Department, Institute of Chemical Technology, Matunga, Mumbai 400 019, India
a r t i c l e
i n f o
Article history: Available online 8 December 2012 Keywords: Cocoa butter alternative Kokum kernel fat Extraction Three phase partitioning
a b s t r a c t Kokum kernel is a byproduct of agro-processing industry in India containing about 40–50% fat which has the potential as a worthy cocoa butter alternative (CBA). However, inefficient extraction techniques that are practiced at cottage level restrict its industrial applications. This work reports on the optimization of the technique of three phase partitioning (TPP) for efficient extraction of kokum kernel fat. The parameters of TPP were optimized with respect to ammonium sulphate concentration, ratio of slurry to t-butanol and pH of slurry. The optimized protocol resulted in maximum recovery of 95% (w/w) fat recovery within 2 h. The technique is economical and eco-friendly, and is promising for utilization of agro-processing waste in India to a product of commercial significance. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Chocolates command an enviable position among food products due to its taste and physical attributes such as snap and the fast and complete melting in the mouth (Lipp and Anklam, 1998a). Major ingredients of chocolate like sugar, milk solids and cocoa powder are distributed evenly in continuous phase of cocoa butter. Cocoa butter triacylglycerols have saturated fatty acids at the 1, 3-positions and oleic acid at the 2-position with oleic (35%), stearic (34%) and palmitic acid (26%) as the main fatty acids (Talbot, 1999). The unique physico-chemical properties of cocoa butter contribute to the desired and characteristic sensory perception of chocolate. There are intensive efforts to replace cocoa butter in chocolate production for technological reasons such as low temperature resistance in hot tropical climates, fat bloom, and higher tempering time, and also for economic reasons since cocoa butter is an expensive commodity with a wide range of price fluctuation. Such cocoa butter alternatives (CBA) are vegetable fats and can consist of palm and palm kernel oil, illipe fat, shea butter, sal fat, kokum kernel fat and mango kernel fat (Lipp and Anklam, 1998a). Kokum (Garcinia indica) is a small, slender evergreen tree found in several parts of India (Maheshwari and Yella Reddy, 2005). The ripe kokum fruit is red or dark purple colored containing 5–8 large seeds. Kokum seeds are solid wastes obtained from kokum processing industry, and contain about 40–50% fat. At present, India produces 10,200 tons of kokum fruit per year (Kshirsagar, 2008), which has the potential to yield about 1000 tons of fat. Crude
⇑ Corresponding author. Tel.: +91 22 3361 2512; fax: +91 22 3361 1020. E-mail address: [email protected] (R.S. Singhal). 0260-8774/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jfoodeng.2012.10.051
kokum kernel fat is reported to be a light yellow hard solid with a faint odour, an iodine value of 35–37, and a saponification value of 189 (Raju and Reni, 2001). It has a melting point of 39–42 °C with major fatty acids being stearic (50–60%) and oleic (36–40%); the major triacylglycerols are 2-oleodistearin (SOS), present to the extent of about 70% suggesting its potential to become a worthy CBE (Reddy and Prabhakar, 1994). Stearic acid and stearic acid rich acylglycerols are absorbed less efficiently than lauric, myristics and palmitic acids (Kritchevsky, 1994). Hence, kokum fat high in stearic acid and low in palmitic acid is less atherogenic than other CBA containing higher palmitic acid contents, and can be a healthy source of CBA in chocolate and confectionary products. Conventionally kokum fat is obtained at a cottage level by crushing the kernels, boiling in water, and skimming the fat from the top. This technique gives a poor recovery of about 25–31% fat. This is the major constraint in extraction of kokum fat at commercial scale. Fat is also obtained by multistage solvent extraction with hexane, but it is economical only when done at large scale. Hexane is used extensively for solvent extraction of edible oil, but it is flammable and non-biorenewable (Gandhi et al., 2003). Hexane emitted from oilseed extraction is a source of volatile organic compounds. It has been identified as an air pollutant since it can react with other pollutants to produce ozone and photochemical oxidants (Wan et al., 1995; Ferreira-Dias et al., 2003). Hence an efficient, economical and eco-friendly technique is required for extraction of fat from kokum seeds. Three phase partitioning (TPP) is a simple novel bioseparation and purification technique in which a salt (e.g. ammonium sulphate) and water miscible aliphatic alcohol (e.g. t-butanol) are added to an aqueous solution containing proteins (Roy et al., 2005). Under optimized conditions, three phases are formed within an hour.
465
G.S. Vidhate, R.S. Singhal / Journal of Food Engineering 117 (2013) 464–466
Pigments, lipids and enzyme inhibitors are concentrated in the upper solvent phase which is separated from lower aqueous phase enriched with polar components like saccharides by an intermediate protein precipitated layer (Kiss et al., 1998). TPP has been extensively evaluated for simultaneous separation and purification of proteins, enzymes and inhibitors from crude suspensions. The physiochemical basis of TPP is quite complex and is believed to involve ionic strength effects, kosmotropy, cavity surface tension enhancement, osmotic stress, and exclusion-crowding effects (Roy et al., 2005). t-Butanol increases the buoyancy of the precipitated protein by binding to it which results in its floatation above the denser aqueous salt layer (Rajeeva and Lele, 2011). TPP has been evaluated for extraction of oleaginous material from soybeans (Sharma et al., 2002) and Jatropha curcas L. (Shah et al., 2004). TPP requires less time, an efficiency that is comparable to Soxhlet extraction, enables working at room temperature, permits recycling of the chemicals, is easily scalable, and shows rapid recovery (Kiss et al., 1998). The present work was planned to optimize the conditions of TPP for maximum recovery of the fat from kokum seeds with use of more safe solvents than hexane. 2. Materials and methods 2.1. Materials Kokum fruits of Garcinia indica Choisy variety were purchased from Ratnagiri, Maharashtra, India. Ammonium sulphate (CAS-No. 7783-20-2), petroleum ether 60–80 °C (CAS-No. 8032-32-4), ethanol (CAS-No. 64-17-5) and iso-propanol (CAS-No. 67-63-0) were obtained from S.D. Fine Chemicals Limited, Mumbai, India. t-Butanol (CAS-No. 75-65-0) was obtained from Thomas Baker, Mumbai, India. 2.2. Methods 2.2.1. Total fat content in kokum kernel powder Kokum seeds were separated from matured kokum fruits manually, and washed with hot water 3 to 4 times to remove gummy material adhered to seeds. These seeds were then dried in a tray dryer at 70 °C to reduce the moisture content from an initial value of 27% to below 10%. The loosened shells were separated manually to obtain kernels. Dried kokum kernels were powdered in hand grinder (Mill A 11 basic Analytical mill, IKAÒ, India) to get fine powder of 1000 lm which would aid the extraction. The total fat in kokum kernel powder was determined by Soxhlet extraction using petroleum ether as a solvent as per the standard AOAC Official method 920.39-4.5.01. 2.2.2. Three phase partitioning Slurry was prepared by dispersing 1 g kokum kernel powder in 16 ml distilled water by gentle stirring on a magnetic stirrer. The pH of slurry was noted and then adjusted to the desired value by adding 0.1 N HCl or 0.1 N NaOH. Weighed amount of ammonium sulphate (10 to 60% w/v of slurry) was added to the slurry prepared and vortexed gently, followed by addition of measured amount of t-butanol (t-butanol to slurry ratio = 0.5:1 to 3:1). This slurry of salt
and solvent system was mixed properly by gentle stirring on magnetic stirrer for 30 min. In order to form three phases, system was allowed to stand at 45 °C in a water bath for 1 h. The three phases formed were separated by centrifugation at 2900g for 10 min at 30 °C. The upper organic layer of t-butanol containing extracted fat was collected and the t-butanol was evaporated on a rotary evaporator to obtain the extracted fat. The amounts of fat recovered by Soxhlet extraction was considered as 100% while calculating amount of fat recovered by TPP. One factor at- a- time method was used to study effect of different parameters and is detailed in Table 1. All extractions were carried out in triplicates. 3. Results and discussion The total fat content in kokum kernel was found to be 49 (% w/ w) by Soxhlet extraction using petroleum ether as the solvent. This fat recovery was considered as 100 (% w/w) for calculating oil recovery by TPP. Ammonium sulphate concentration, t-butanol to slurry volume ratio, and pH of slurry were found to be critical parameters for evaluation of TPP and therefore significant to obtain maximum fat recovery. Selective and efficient precipitation of protein depends upon the concentration of ammonium sulphate salt. Hence it was optimized to precipitate maximum protein in intermediate layer in TPP, which further implies the maximum separation of the fat in the organic phase. The extraction of fat increased with an increase in salt concentration up to 50 (% w/v) (Fig. 1a). The increment in fat recovery was not statistically significant at salt concentration of 60 (% w/v) (Student’s t-test, p > 0.05). Hence, 50 (% w/v) ammonium sulphate was considered to be sufficient to get maximum recovery of 94.56 (% w/w). Another important parameter for TPP was found to be t-butanol to slurry ratio (v/v) (Fig. 1b). At lower content of t-butanol (t-butanol to slurry ratio = 0.5:1), it may not adequately synergize with ammonium sulphate (Sharma and Gupta, 2001). Whereas no statistically significant increase (Student’s t-test, p > 0.05) in the fat recovery was observed when the ratio of t-butanol to slurry was increased from 1:1 to 3:1. This could be due to high t-butanol content, which may cause denaturation of the protein and hinders protein precipitation (Chaiwut et al., 2010). Hence a ratio of 1:1 was considered as optimum with resulting fat recovery of 94.22 (% w/w). The basic mechanism of TPP is based on the binding of sulphate anion to the cationic sites in the proteins (Roy et al., 2005). This is significantly influenced by pH of the slurry. The pH should be kept lower than the pI of the target proteins (Roy et al., 2005), as proteins are positively charged and quantitatively precipitated out by TPP (Rajeeva and Lele, 2011). However, in certain cases, it has been found that TPP works better at pH above pI of the protein (Roy et al., 2005). Since the precipitation of protein is linked to efficient separation of fat constituents from crude aqueous solutions phase to organic phase, the pH of the aqueous slurry was varied from acidic to alkaline conditions for the present system (Fig. 1c). Maximum fat recovery of 95.37 (% w/w) was obtained at an acidic pH of 2.0.t-Butanol was replaced with iso-propanol and ethanol as solvent in TPP, but t-butanol was found to be better (Fig. 1d). This
Table 1 Evaluation of parameters used for three phase partitioning. Variable parameter (range)
Salt (% w/v) (10 to 60) Ratio (0.5:1, 1:1, 2:1, 3:1) pH (2,4,7,9) Solvent (t-butanol, IPA, ethanol)
Constant parameters Salt (% w/v)
Ratio
pH
Solvent
– 50 50 50
1:1 – 1:1 1:1
Slurry pH (2.3) Slurry pH (2.3) – 2
t-Butanol t-Butanol t-Butanol –
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G.S. Vidhate, R.S. Singhal / Journal of Food Engineering 117 (2013) 464–466
c)
100.00
100.00
Fat Recovery (% w/w)
Fat Recovery (% w/w)
a)
80.00 60.00 40.00 20.00
80.00 60.00 40.00 20.00 0.00
0.00 10
20
30
40
50
60
2
Ammonium sulphate concentration (% w/v)
b)
4
7
9
pH
d)
100.00
Fat Recovery (% w/w)
Fat Recovery (% w/w)
100.00 80.00 60.00 40.00 20.00 0.00
80.00 60.00 40.00 20.00 0.00
0.5:1
1:1 2:1 t-Butanol to slurry ratio (v/v)
3:1
t-Butanol
Isopropanol
Ethanol
Fig. 1. (a) Effect of varying amount of ammonium sulphate concentration, (b) Effect of varying the ratio of slurry to t-butanol, (c) Effect of varying pH of slurries, and (d) Effect of different solvents on fat recovery.
could be due to less solubility of oil in ethanol or IPA. t-Butanol has a higher boiling point (84 °C) than hexane (69 °C) (Sharma et al., 2002), so solvent release from closed system to atmosphere will be very less and therefore TPP with t-butanol is more eco-friendly than hexane. Separation of t-butanol (freezing point of 11 °C) by chilling is more economical than separation by heating, as chilling requires less energy than evaporation (Gaur et al., 2007). 4. Conclusion A maximum recovery of 95 (% w/w) fat was obtained from kokum kernels with evaluated TPP system consisting of 50 (% w/v) salt concentration, 1:1 ratio of slurry to t-butanol, and a pH of 2.0 within 2 h. Exploiting this technology could mobilize agroindustrial wastes such as kokum kernel for value added ingredients for food and cosmetic industries. It is envisaged that extensive mathematical modeling, pilot plant trials and industrial scale-up are urgently required before it can be recommended for use at commercial level. Acknowledgments The authors would like to thank University Grants Commission, Government of India for their financial support. References Chaiwut, P., Pintathong, P., Rawdkuen, S., 2010. Extraction and three-phase partitioning behavior of proteases from papaya peels. Process Biochemistry 45, 1172–1175. Ferreira-Dias, S., Valente, D.G., Abreu, J.M.F., 2003. Comparison between ethanol and hexane for oil extraction from Quercus suber L. fruits. Grasas y Aceites 54, 378– 383. Gandhi, A.P., Joshi, K.C., Jha, K., Parihar, V.S., Srivastav, D.C., Raghunadh, P., Kawalkar, J., Jain, S.K., Tripathi, R.N., 2003. Studies on alternative solvents for the
extraction of oil-I soybean. International Journal of Food Science and Technology 38, 369–375. Gaur, R., Sharma, A., Khare, S.K., Gupta, M.N., 2007. A novel process for extraction of edible oils: Enzyme assisted three phase partitioning (EATPP). Bioresource Technology 98, 696–699. Kiss, E., Szamos, J., Tamas, B., Borbas, R., 1998. Interfacial behavior of proteins in three-phase partitioning using salt-containing water/tert-butanol systems. Colloids and Surfaces A: Physicochemical and Engineering Aspects 142, 295– 302. Kritchevsky, D., 1994. Stearic acid metabolism and atherogenesis: history. American Journal of Clinical Nutrition 60, 997S–1001S. Kshirsagar, P.J. 2008. Production, processing and marketing of Kokum (Garcinia indica) in Konkan region of Maharashtra – an economic analysis. PhD thesis, University of Agricultural Sciences, Dharwad, India. Lipp, M., Anklam, E., 1998. Review of cocoa butter and alternative fats for use in chocolate-part A. compositional data. Food Chemistry 62, 73–97. Maheshwari, B., Yella Reddy, S., 2005. Application of kokum (Garcinia indica) fat as cocoa butter improver in chocolate. Journal of the Science of Food and Agriculture 85, 135–140. Rajeeva, S., Lele, S.S., 2011. Three-phase partitioning for concentration and purification of laccase produced by submerged cultures of Ganoderma sp. WR1. Biochemical Engineering Journal 54, 103–110. Raju, V.K., Reni, M., 2001. Kokam and cambodge. In: Peter, K.V. (Ed.), Handbook of Herbs and Spices. Woodhead Publishing Limited, Cambridge, pp. 207–215. Reddy, S.Y., Prabhakar, J.V., 1994. Cocoa butter extenders from kokum (Garcinia indica) and phulwara (Madhuca butyracea) butter. Journal of the American Oil Chemists Society 71, 217–219. Roy, I., Sharma, A., Gupta, M.N., 2005. Recovery of biological activity in reversibly inactivated proteins by three phase partitioning. Enzyme and Microbial Technology 37, 113–120. Shah, S., Sharma, A., Gupta, M.N., 2004. Extraction of oil from Jatropha curcas L. seed kernels by enzyme assisted three phase partitioning. Industrial Crops and Products 20, 275–279. Sharma, A., Gupta, M.N., 2001. Three phase partitioning as a large-scale separation method for purification of a wheat germ bifunctional protease/amylase inhibitor. Process Biochemistry 37, 193–196. Sharma, A., Khare, S.K., Gupta, M.N., 2002. Three phase partitioning for extraction of oil from soybean. Bioresource Technology 85, 327–329. Talbot, G., 1999. Chocolate temper. In: Beckett, S.T. (Ed.), Industrial Chocolate Manufacture and Use. Blackwell Science, Oxford, pp. 218–230. Wan, P.J., Pakarinen, D.R., Hron, R.J., Richard, O.L., Conkerton, E.J., 1995. Alternative hydrocarbon solvents for cottonseed extraction. Journal of the American Oil Chemists Society 72, 661–664.
Contents lists available at SciVerse ScienceDirect
Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Extraction of cocoa butter alternative from kokum (Garcinia indica) kernel by three phase partitioning Ganesh S. Vidhate, Rekha S. Singhal ⇑ Food Engineering and Technology Department, Institute of Chemical Technology, Matunga, Mumbai 400 019, India
a r t i c l e
i n f o
Article history: Available online 8 December 2012 Keywords: Cocoa butter alternative Kokum kernel fat Extraction Three phase partitioning
a b s t r a c t Kokum kernel is a byproduct of agro-processing industry in India containing about 40–50% fat which has the potential as a worthy cocoa butter alternative (CBA). However, inefficient extraction techniques that are practiced at cottage level restrict its industrial applications. This work reports on the optimization of the technique of three phase partitioning (TPP) for efficient extraction of kokum kernel fat. The parameters of TPP were optimized with respect to ammonium sulphate concentration, ratio of slurry to t-butanol and pH of slurry. The optimized protocol resulted in maximum recovery of 95% (w/w) fat recovery within 2 h. The technique is economical and eco-friendly, and is promising for utilization of agro-processing waste in India to a product of commercial significance. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Chocolates command an enviable position among food products due to its taste and physical attributes such as snap and the fast and complete melting in the mouth (Lipp and Anklam, 1998a). Major ingredients of chocolate like sugar, milk solids and cocoa powder are distributed evenly in continuous phase of cocoa butter. Cocoa butter triacylglycerols have saturated fatty acids at the 1, 3-positions and oleic acid at the 2-position with oleic (35%), stearic (34%) and palmitic acid (26%) as the main fatty acids (Talbot, 1999). The unique physico-chemical properties of cocoa butter contribute to the desired and characteristic sensory perception of chocolate. There are intensive efforts to replace cocoa butter in chocolate production for technological reasons such as low temperature resistance in hot tropical climates, fat bloom, and higher tempering time, and also for economic reasons since cocoa butter is an expensive commodity with a wide range of price fluctuation. Such cocoa butter alternatives (CBA) are vegetable fats and can consist of palm and palm kernel oil, illipe fat, shea butter, sal fat, kokum kernel fat and mango kernel fat (Lipp and Anklam, 1998a). Kokum (Garcinia indica) is a small, slender evergreen tree found in several parts of India (Maheshwari and Yella Reddy, 2005). The ripe kokum fruit is red or dark purple colored containing 5–8 large seeds. Kokum seeds are solid wastes obtained from kokum processing industry, and contain about 40–50% fat. At present, India produces 10,200 tons of kokum fruit per year (Kshirsagar, 2008), which has the potential to yield about 1000 tons of fat. Crude
⇑ Corresponding author. Tel.: +91 22 3361 2512; fax: +91 22 3361 1020. E-mail address: [email protected] (R.S. Singhal). 0260-8774/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jfoodeng.2012.10.051
kokum kernel fat is reported to be a light yellow hard solid with a faint odour, an iodine value of 35–37, and a saponification value of 189 (Raju and Reni, 2001). It has a melting point of 39–42 °C with major fatty acids being stearic (50–60%) and oleic (36–40%); the major triacylglycerols are 2-oleodistearin (SOS), present to the extent of about 70% suggesting its potential to become a worthy CBE (Reddy and Prabhakar, 1994). Stearic acid and stearic acid rich acylglycerols are absorbed less efficiently than lauric, myristics and palmitic acids (Kritchevsky, 1994). Hence, kokum fat high in stearic acid and low in palmitic acid is less atherogenic than other CBA containing higher palmitic acid contents, and can be a healthy source of CBA in chocolate and confectionary products. Conventionally kokum fat is obtained at a cottage level by crushing the kernels, boiling in water, and skimming the fat from the top. This technique gives a poor recovery of about 25–31% fat. This is the major constraint in extraction of kokum fat at commercial scale. Fat is also obtained by multistage solvent extraction with hexane, but it is economical only when done at large scale. Hexane is used extensively for solvent extraction of edible oil, but it is flammable and non-biorenewable (Gandhi et al., 2003). Hexane emitted from oilseed extraction is a source of volatile organic compounds. It has been identified as an air pollutant since it can react with other pollutants to produce ozone and photochemical oxidants (Wan et al., 1995; Ferreira-Dias et al., 2003). Hence an efficient, economical and eco-friendly technique is required for extraction of fat from kokum seeds. Three phase partitioning (TPP) is a simple novel bioseparation and purification technique in which a salt (e.g. ammonium sulphate) and water miscible aliphatic alcohol (e.g. t-butanol) are added to an aqueous solution containing proteins (Roy et al., 2005). Under optimized conditions, three phases are formed within an hour.
465
G.S. Vidhate, R.S. Singhal / Journal of Food Engineering 117 (2013) 464–466
Pigments, lipids and enzyme inhibitors are concentrated in the upper solvent phase which is separated from lower aqueous phase enriched with polar components like saccharides by an intermediate protein precipitated layer (Kiss et al., 1998). TPP has been extensively evaluated for simultaneous separation and purification of proteins, enzymes and inhibitors from crude suspensions. The physiochemical basis of TPP is quite complex and is believed to involve ionic strength effects, kosmotropy, cavity surface tension enhancement, osmotic stress, and exclusion-crowding effects (Roy et al., 2005). t-Butanol increases the buoyancy of the precipitated protein by binding to it which results in its floatation above the denser aqueous salt layer (Rajeeva and Lele, 2011). TPP has been evaluated for extraction of oleaginous material from soybeans (Sharma et al., 2002) and Jatropha curcas L. (Shah et al., 2004). TPP requires less time, an efficiency that is comparable to Soxhlet extraction, enables working at room temperature, permits recycling of the chemicals, is easily scalable, and shows rapid recovery (Kiss et al., 1998). The present work was planned to optimize the conditions of TPP for maximum recovery of the fat from kokum seeds with use of more safe solvents than hexane. 2. Materials and methods 2.1. Materials Kokum fruits of Garcinia indica Choisy variety were purchased from Ratnagiri, Maharashtra, India. Ammonium sulphate (CAS-No. 7783-20-2), petroleum ether 60–80 °C (CAS-No. 8032-32-4), ethanol (CAS-No. 64-17-5) and iso-propanol (CAS-No. 67-63-0) were obtained from S.D. Fine Chemicals Limited, Mumbai, India. t-Butanol (CAS-No. 75-65-0) was obtained from Thomas Baker, Mumbai, India. 2.2. Methods 2.2.1. Total fat content in kokum kernel powder Kokum seeds were separated from matured kokum fruits manually, and washed with hot water 3 to 4 times to remove gummy material adhered to seeds. These seeds were then dried in a tray dryer at 70 °C to reduce the moisture content from an initial value of 27% to below 10%. The loosened shells were separated manually to obtain kernels. Dried kokum kernels were powdered in hand grinder (Mill A 11 basic Analytical mill, IKAÒ, India) to get fine powder of 1000 lm which would aid the extraction. The total fat in kokum kernel powder was determined by Soxhlet extraction using petroleum ether as a solvent as per the standard AOAC Official method 920.39-4.5.01. 2.2.2. Three phase partitioning Slurry was prepared by dispersing 1 g kokum kernel powder in 16 ml distilled water by gentle stirring on a magnetic stirrer. The pH of slurry was noted and then adjusted to the desired value by adding 0.1 N HCl or 0.1 N NaOH. Weighed amount of ammonium sulphate (10 to 60% w/v of slurry) was added to the slurry prepared and vortexed gently, followed by addition of measured amount of t-butanol (t-butanol to slurry ratio = 0.5:1 to 3:1). This slurry of salt
and solvent system was mixed properly by gentle stirring on magnetic stirrer for 30 min. In order to form three phases, system was allowed to stand at 45 °C in a water bath for 1 h. The three phases formed were separated by centrifugation at 2900g for 10 min at 30 °C. The upper organic layer of t-butanol containing extracted fat was collected and the t-butanol was evaporated on a rotary evaporator to obtain the extracted fat. The amounts of fat recovered by Soxhlet extraction was considered as 100% while calculating amount of fat recovered by TPP. One factor at- a- time method was used to study effect of different parameters and is detailed in Table 1. All extractions were carried out in triplicates. 3. Results and discussion The total fat content in kokum kernel was found to be 49 (% w/ w) by Soxhlet extraction using petroleum ether as the solvent. This fat recovery was considered as 100 (% w/w) for calculating oil recovery by TPP. Ammonium sulphate concentration, t-butanol to slurry volume ratio, and pH of slurry were found to be critical parameters for evaluation of TPP and therefore significant to obtain maximum fat recovery. Selective and efficient precipitation of protein depends upon the concentration of ammonium sulphate salt. Hence it was optimized to precipitate maximum protein in intermediate layer in TPP, which further implies the maximum separation of the fat in the organic phase. The extraction of fat increased with an increase in salt concentration up to 50 (% w/v) (Fig. 1a). The increment in fat recovery was not statistically significant at salt concentration of 60 (% w/v) (Student’s t-test, p > 0.05). Hence, 50 (% w/v) ammonium sulphate was considered to be sufficient to get maximum recovery of 94.56 (% w/w). Another important parameter for TPP was found to be t-butanol to slurry ratio (v/v) (Fig. 1b). At lower content of t-butanol (t-butanol to slurry ratio = 0.5:1), it may not adequately synergize with ammonium sulphate (Sharma and Gupta, 2001). Whereas no statistically significant increase (Student’s t-test, p > 0.05) in the fat recovery was observed when the ratio of t-butanol to slurry was increased from 1:1 to 3:1. This could be due to high t-butanol content, which may cause denaturation of the protein and hinders protein precipitation (Chaiwut et al., 2010). Hence a ratio of 1:1 was considered as optimum with resulting fat recovery of 94.22 (% w/w). The basic mechanism of TPP is based on the binding of sulphate anion to the cationic sites in the proteins (Roy et al., 2005). This is significantly influenced by pH of the slurry. The pH should be kept lower than the pI of the target proteins (Roy et al., 2005), as proteins are positively charged and quantitatively precipitated out by TPP (Rajeeva and Lele, 2011). However, in certain cases, it has been found that TPP works better at pH above pI of the protein (Roy et al., 2005). Since the precipitation of protein is linked to efficient separation of fat constituents from crude aqueous solutions phase to organic phase, the pH of the aqueous slurry was varied from acidic to alkaline conditions for the present system (Fig. 1c). Maximum fat recovery of 95.37 (% w/w) was obtained at an acidic pH of 2.0.t-Butanol was replaced with iso-propanol and ethanol as solvent in TPP, but t-butanol was found to be better (Fig. 1d). This
Table 1 Evaluation of parameters used for three phase partitioning. Variable parameter (range)
Salt (% w/v) (10 to 60) Ratio (0.5:1, 1:1, 2:1, 3:1) pH (2,4,7,9) Solvent (t-butanol, IPA, ethanol)
Constant parameters Salt (% w/v)
Ratio
pH
Solvent
– 50 50 50
1:1 – 1:1 1:1
Slurry pH (2.3) Slurry pH (2.3) – 2
t-Butanol t-Butanol t-Butanol –
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G.S. Vidhate, R.S. Singhal / Journal of Food Engineering 117 (2013) 464–466
c)
100.00
100.00
Fat Recovery (% w/w)
Fat Recovery (% w/w)
a)
80.00 60.00 40.00 20.00
80.00 60.00 40.00 20.00 0.00
0.00 10
20
30
40
50
60
2
Ammonium sulphate concentration (% w/v)
b)
4
7
9
pH
d)
100.00
Fat Recovery (% w/w)
Fat Recovery (% w/w)
100.00 80.00 60.00 40.00 20.00 0.00
80.00 60.00 40.00 20.00 0.00
0.5:1
1:1 2:1 t-Butanol to slurry ratio (v/v)
3:1
t-Butanol
Isopropanol
Ethanol
Fig. 1. (a) Effect of varying amount of ammonium sulphate concentration, (b) Effect of varying the ratio of slurry to t-butanol, (c) Effect of varying pH of slurries, and (d) Effect of different solvents on fat recovery.
could be due to less solubility of oil in ethanol or IPA. t-Butanol has a higher boiling point (84 °C) than hexane (69 °C) (Sharma et al., 2002), so solvent release from closed system to atmosphere will be very less and therefore TPP with t-butanol is more eco-friendly than hexane. Separation of t-butanol (freezing point of 11 °C) by chilling is more economical than separation by heating, as chilling requires less energy than evaporation (Gaur et al., 2007). 4. Conclusion A maximum recovery of 95 (% w/w) fat was obtained from kokum kernels with evaluated TPP system consisting of 50 (% w/v) salt concentration, 1:1 ratio of slurry to t-butanol, and a pH of 2.0 within 2 h. Exploiting this technology could mobilize agroindustrial wastes such as kokum kernel for value added ingredients for food and cosmetic industries. It is envisaged that extensive mathematical modeling, pilot plant trials and industrial scale-up are urgently required before it can be recommended for use at commercial level. Acknowledgments The authors would like to thank University Grants Commission, Government of India for their financial support. References Chaiwut, P., Pintathong, P., Rawdkuen, S., 2010. Extraction and three-phase partitioning behavior of proteases from papaya peels. Process Biochemistry 45, 1172–1175. Ferreira-Dias, S., Valente, D.G., Abreu, J.M.F., 2003. Comparison between ethanol and hexane for oil extraction from Quercus suber L. fruits. Grasas y Aceites 54, 378– 383. Gandhi, A.P., Joshi, K.C., Jha, K., Parihar, V.S., Srivastav, D.C., Raghunadh, P., Kawalkar, J., Jain, S.K., Tripathi, R.N., 2003. Studies on alternative solvents for the
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