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International Journal of Agricultural and Food Science Universal Research Publications. All rights reserved
ISSN 2249-8516 Original Article Effects of Phospholipase A2 degumming on Palm Oil components Amit Kumar Mukherjee*, Kona Mondal, Mohamed Asif Ikbal Akhan and Suchismita Biswas Department of Food Technology, Haldia Institute of Technology, P. O. HIT, Hatiberia, Purba Medinipur, Haldia – 721657, W. B., India *Corresponding author. E-mail:
[email protected] Tel: + 91 9477290235. Fax: +913224 252800/253062. Received 15 May 2013; accepted 24 May 2013 Abstract Enzyme-aided degumming of palm oil with phospholipase A2 produced degummed oil with reduced free fatty acid content. The free fatty acid content of enzyme-treated oil was 10.72% as against that of crude palm oil, 10.43%. Conventional acid degumming with H3PO4 resulted in a higher free fatty acid content in oil (12.18%). Incubation of crude palm oil with 0.1 mg/kg phospholipase A2 at 50°C and pH 5 not only removed most of the phosphatides (97.14%), but also maintained a higher yield of degummed oil. Enzymatic degumming also induced better colour of crude oil as compared to acid degumming process. Enzymatic process was found to prevent free fatty acid formation in oil in excess to that produced by hydrolysis of phospholipids. © 2013 Universal Research Publications. All rights reserved Key words: palm oil, phospholipids, phospholipase A2, enzymatic degumming, nonhydratable phosphatides. 1. Introduction: Since after its introduction in edible oil processing, enzymatic degumming [1] has been proved to be an effective alternative for conventional chemical degumming process. Oils degummed either with a single type of phospholipase [2] or with a combination of two such enzymes can almost entirely remove phospholipids under suitable conditions of reaction [3]. Phospholipases are group of hydrolases highly specific in their actions on phospholipids. The advantage of using these enzymes in the degumming operation is that they can effectively remove most of the phopsphatides from crude oil thereby reducing the consumption of excess of NaOH over the calculated amount during the subsequent refining operation and minimizing refining loss. Phospholipase A1, A2, C and D all react with nonhydratable phospholipids [4] producing hydratable lysophospholipids containing stoichiometrically at least 50%, and as high as 100% of the initial fatty acids esterified with the glycerol. The enzymatic process of degumming has so far been applied on soybean, sunflower, canola and several other vegetable oils with highly satisfactory results. However, the effect of phospholipases on palm oil has not been studied extensively till date. Phospholipases are present in all living cells and are isolated from plants, higher animals and microorganism. Phospholipase A2 or PLA2 (International Union of Biochemistry and Molecular Biology, IUBMB
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nomenclature 3.1.1.4) is used in industry for production of emulsifiers like lysolecithin and monoglycerides, and for the manufacture of food mayonnaise [5]. PLA2 hydrolyses the ester linkage at sn-2 carbon of glycerophospholipids (Figure 1). Palm oil is extracted from the fleshy mesocarp of oil palm fruit and is characterised by the presence of almost equal amount of saturated (16:0) and unsaturated (18:1) fatty acids in its triglyceride [6]. The presence of palmitic acid is responsible for low iodine value of palm oil. Crude palm oil (CPO) is intensely red coloured due to the presence of carotenoid pigments and the oil partially solidifies below 30°C. The free fatty acid content of palm oil greatly varies depending upon the source. The present work of PLA2 (from porcine pancreas, Sigma Aldrich)aided degumming of palm oil produced highly satisfactory results in terms of total oil yield, amount of phosphorous removed, residual free fatty acids (FFA) in oil and the colour of the final oil. After hydrolysis of glycerophospholipids by PLA2, the lysophospholipid fractions dissolve in aqueous phase and the fatty acids produced remain in the oil phase. PLA2 remains in water phase while catalysing the hydrolysis of oil. In case of acid degumming with phosphoric or citric acid [7], more such free fatty acids are produced due to a high temperature operation (80°-110°C) for prolonged time. 2. Materials and methods: CPO obtained from industry was melted at 40°C to get a
International Journal of Agricultural and Food Science 2013, 3(2): 69-71
Figure 1: Site for action of different phospholipase enzymes. ‘X’ is any amine or sugar group. homogeneous liquid and its free fatty acids, saponification number, iodine value, colour and total phosphorous (mg/kg) were determined following standard methods of analyses. After characterization of crude oil, it was subjected to enzymatic degumming, acid degumming and water degumming successively. 0.1 Kilogram of crude palm oil was taken in each case. The gum-free oil was tested for its free fatty acid content, saponification number, iodine value, colour in photometric scale and phosphorous content. The yield of degummed oil was measured for each set of experiment and the results were compared. 2.1 Degumming of crude palm oil 2.1.1 Phospholipase A2 degumming 0.1 Kilogram of crude oil was taken for enzymatic degumming. A PLA2 dose of 0.1 mg/kg was employed on oil at pH 5 and was incubated at 50°C for 30 minutes. The pH was maintained with 0.1 M citrate buffer of pH 5. The oil containing lysophospholipids was hydrated with 5% (v/v) water at 60°C for 10 minutes. After completion of the reaction, the hydrated gum was removed by centrifugation at 6000 R. P. M. for 15 minutes. 2.1.2 Phosphoric acid degumming The acid degumming of CPO was done with 0.05% phosphoric acid at 80°C for 15 minutes on 0.1 kilogram of crude oil. The residual acid is removed from oil by washing with 5% water at 60°C as above. The aqueous phase containing the hydrated gums was separated from oil by centrifugation (6000 R. P. M., 15 minutes). 2.1.3 Water degumming Water degumming was carried out with distilled water (5%) at 80°C for 15 minutes with the same amount of crude oil as in the earlier cases followed by removal of gum by centrifugation. 2.2 Characterization of crude and processed palm oil 2.2.1 Free fatty acid For estimation of free fatty acids, 0.005 kg of oil sample was titrated with ethanolic potassium hydroxide (KOH) using phenolphthalein as indicator. The amount of FFA was expressed as percentage oleic acid in oil. 2.2.2 Saponification number Saponification number was expressed as number of milligrams of KOH required to saponify 0.001 kg of oil. 2.2.3 Iodine value Iodine value was expressed as the number of grams of iodine absorbed by 0.1 kg of oil. Twenty five millilitres of iodine monochloride (ICl) in glacial acetic acid (Wijs solution) was reacted with 0.001 kg of oil (dissolved in 15 ml carbon tetrachloride) for 30 minutes in the darkness for introduction of halogens quantitatively to the double bonds
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of the fatty acids in triglycerides of oil. The remaining ICl was reacted with 20 millilitres of 10% KI solution for liberation of iodine, which was estimated by titration with standard 0.1 N sodium thiosulphate (Na2S2O3) solution using starch indicator. A blank without oil was estimated for total iodine in 25 ml iodine monochloride solution. 2.2.4 Photometric colour The colour of oil was measured on photometric scale [8] by measuring the absorbance at different wavelengths with a spectrophotometer and by putting the values in the standard equation. 2.2.5 Phosphorous content The amount of phosphorous in oil was estimated by molybdenum blue method [9] by decomposition of organic components in palm oil with zinc oxide followed by ignition in a muffle furnace at 550°C and dissolving the ash in acid. The results obtained in mg of phosphorous per kg of oil were expressed as percentage of total phosphorous of crude palm oil removed after enzymatic, acid and water degumming. 3. Results and discussion The CPO, on PLA2 degumming, produced an oil with negligible amount of residual phosphorous (2.86%) as indicated in Table 1. The enzymatic process was found to be efficient in controlling the FFA (10.72%) formation in oil. The FFA content of PLA2 treated oil was also found to be lower as compared to that of both acid degummed (12.18%) and water degummed oil (11.25%). The effects of enzyme, acid and water treatments on CPO are furnished in Figure 2 as percentage FFA, colour and percentage residual phosphorous in treated oil. The yield of enzyme-treated oil was also found to be higher (95 - 96%) as compared to other two processes (93 - 94%). No significant change in iodine value was observed after any of the above three degumming operations. The iodine values of oil after enzymatic degumming, acid degumming and water degumming were 54.69, 54.5 and 54.6 respectively. The CPO had an iodine value of 55.22.
Figure 2: Percentage FFA, iodine value, colour and percentage residual phosphorous in oil after PLA2 degumming, phosphoric acid degumming and water degumming.
The palm oil is characterised by its high amount of NHP and simple water degumming operation is not effective in removal of its phosphatides. The presence of bivalent metal ions like Ca++, Mg++ and Fe++ are responsible for non-polar behaviour [10] of palm oil phosphatides. The non-polar nature and difficulty in removing the NHP by water International Journal of Agricultural and Food Science 2013, 3(2): 69-71
degumming before physical refining, made palm oil phospholipids a major concern for oil producers. Enzymatic
degumming improved colour without any reduction in degree of unsaturation of the original oil.
Table 1: Analysis of crude, enzyme-treated, acid-treated and water degummed palm oil. Parameter CPO PLA2 degumming H3PO4 degumming FFA (%) Saponification number Iodine value Photometric colour Phosphorous removal (%) Yield of oil (%)
10.43 246.84 55.22 194.17 _ _
10.72 243.1 54.69 141.2 97.14 95-96
4. Conclusions Today around 44% of total oils and fats exported throughout the world is palm oil [11]. The physical refining of palm oil is difficult due to its significantly high level of phosphatides (2-5%) in many species of oil palm fruit. The presence of gum in oil imparts higher loss during refining and reduces storage life of oil. Enzymatic process is a better alternative to conventional high temperature chemical degumming process. This not only reduces the costs of steam and chemical consumption, but also gives a better yield of good quality oil. One disadvantage of the enzymatic process is the high cost of enzyme. If this disadvantage is successfully overcome, phospholipase degumming can entirely replace the chemical degumming operation in the coming days. Abbreviations: PLA2 – Phospholipase A2, CPO – Crude palm oil, NHPNonhydratable phosphatides, FFA – Free fatty acids. References: 1. F. D. Gunstone, The Lipid Handbook, second ed., Chapman and Hall, London, 1994. 2. S. Dixit, S. Kanakraj. Enzymatic degumming of feedstock’s (vegetable oil) for bio-diesel – a review, J. Engg. Sc. & Manage. edu. 3 (2010) 57-59. 3. C. L. G. Dayton, E. M. Rosswurm and F. D. S. Galhardo. Enzymatic degumming utilizing a mixture
Water degumming
12.18 244.2 54.5 144.5 95.79 93-94
11.25 245.8 54.6
144.92 77.14 93-94
of PLA and PLC phospholipases with reduced reaction time, U. S. Patent US 0069587, 2009. 4. J. G. Yang, Y. Wang, B. Yang, G. Mainda, Y. Guo, Degumming of vegetable oil by a new microbial lipase, Food Technol. Biotechnol. 44 (2006) 101-104. 5. L. D. Maria, J. Vind, K. M. OxenbØll, A. Svendsen, Phospholipases and their Industrial Applications, Appl. Microbiol. Biotechnol. 74 (2007) 290-300. 6. Y. Basiron, Palm oil, in: F. Shahidi (Eds.), Bailey’s Industrial Oil and Fat products, John Wiley & Sons Inc., 2005, pp. 333-344. 7. L.L.Diosady, P. Sleggs, T. Kaji, Chemical degumming of canola oils, J.Am.Oil Chem. Soc.,59(1982) 313-316. 8. M. M. Chakrabarty, Chemistry and Technology of oils and Fats, Allied Publishers Pvt. Ltd., New Delhi, 2003. 9. E. A. Tosi, A. F. Cazzoli, L. M. Tapiz, Phosphorus in oil. Production of molybdenum blue derivative at ambient temperature using noncarcinogenic reagents, J. Am. Oil Chem. Soc., 75 (1998), 41-44. 10. D. Firestone (Editor), Official Method Ca 12- 55, in: American Oil Chemists’ Society, Champaign, 1989, pp. 1–2. 11. F. D. Gunstone, Production and trade of vegetable oils, in: F. D. Gunstone (Eds.), Vegetable Oils in Food Technology: Composition, Properties and Uses, second ed., Blackwell Publishing Ltd., UK, 2011, pp.1-8.
Source of support: Nil; Conflict of interest: None declared
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International Journal of Agricultural and Food Science 2013, 3(2): 69-71
