De – Acidification Plant for Higher FFA Oils:

Enzymatic deacidification for the production of edible grade triacylglycerols (TG) using high FFA RBO attracts considerable attention for simplicity of the process and no side products. Recycling of enzyme for bio refining of high FFA RBO in laboratory scale as well as in pilot scale is of considerable importance regarding cost effectiveness and time economization. Rice bran (Oryza sativa L) oil may contain 30-40% free fatty acids, if the bran is not processed properly prior to the extraction of oil. High FFA content is one of the main drawbacks in refining RBO due to greater oil loss and darkening of colour [1]. Conventionally, RBO can be alkali refined or physically refined though both methods involve substantial loss of oil. Lipase catalyzed bio refining method may be suitable for RBO of high quality with minimum loss. Moreover, in this process, enzyme can be recycled many more times due to easy isolation of enzyme from the reaction mixture with same efficiency. Productivity of the process can only be obtained by recycling the enzyme which ultimately reduces process cost. The method of deacidification of a vegetable oil consists of conversion of its free fatty acids into neutral glycerides by re esterification with or without the presence of catalyst [2]. Various researchers re esterified high FFA oils using different chemical and biological catalyst. Bhattacharyya and Bhattacharyya [3] deacidified RBO containing 15-30% FFA to 2-3% levels by re esterification with glycerol with or without catalyst. Enzymatic esterification process has been reported by various authors. According to them, FFA content of oil can be reduced to varying level (2-3%) depending on the composition of oil, temperature, nature and concentration of enzymes. Sengupta and Bhattacharyya [4] bio refined high FFA RBO with the help of 1, 3- specific Mucor miehei lipase followed by alkali refining, bleaching and deodorization. They concluded that the high FFA RBO could be refined with a high degree of economy by a combination of enzymatic deacidification and alkali neutralization. Kosugi et al. [5] utilized RBO containing 30-50% FFA to oil containing more than 75% TG by means of immobilized lipase at 60°C for 24 h with dehydration by dry nitrogen flow under a positive nitrogen atmosphere. Kurashige [6] used diacylglycerol (DG) for the esterification of crude palm olein by using a lipase from Pseudomonoas fluorescens. The extent of esterification was high due to better solubility of DG in oil. It was previously reported [7] that monoglyceride (MG) too can esterify FFA. Using this concept, Sengupta and Bhattacharyya [8] bio refined rice bran oil with the help of MG and concluded that MG could be effectively utilized instead of glycerol to reduce FFA producing oil of better quality. But no literatures are obtained regarding the recycling of enzyme for bio refining process of high FFA RBO. Based on this situation, the present study investigated recycling of a non-specific enzyme (Novozyme 435) for deacidification of high FFA RBO up to sixty times in laboratory scale and ten times in pilot scale. By this approach, productivity of the bio refining process can be gained since reusing or recycling the enzyme can reduce process cost. So the efficiency of the process can be enhanced by this way after careful separation of the enzyme from the reaction mixture and by reuses it. This bio refining oil is then stream stripped for making it food grade and by this method, bio refining method can be utilised successfully in large industrial scale also. Experimental

Materials & Methods:

High FFA RBO oil was provided by M/s. Sethia Oils Ltd., Burdwan, West Bengal, India. The enzymes used in the following studies are: TL IM: (Immobilized lipase from Thermomyces lanuginosus with catalytic activity 75 interesterification unit NOVO/gm (IUN/gm) and Novozyme 435 (Candida antarctica) Immobilised lipase. All the enzymes were kind gift of Novozyme South Asia Pvt. Ltd. Bangalore, India. Hexane (B.P. 65- 70°C), diethyl ether (B.P. 35-40°C) and Silica gel G were purchased from S.D. Fine Chemicals (Mumbai, India). Except otherwise specified all other chemicals used were A.R. Grade. Acid value and unsaponifiable of RBO were determined according to standard method described in the official and tentative methods of American Oil Chemists’ Society (1991). Oryzanol content was determined according to the method of Gopala Krishna et al [9]. Degumming, de waxing and bleaching: The high FFA RBO was degummed, de waxed and bleached before bio refining. The oil was degummed by mixing together with 0.25% phosphoric acid of 85% concentration and 2% water on the weight of the oil at 70 °C for 30 min with constant stirring. The gummy materials were separated by centrifugation at 7000 rpm and finally water washed to remove the phosphoric acid. The water washed oil was dried under vacuum at about 90°C. The oil was de waxed by winterisation process at 10-12oC for 7 h followed by centrifugation. The de waxed oil was bleached with 2% by weight of bleaching earth and 0.5% activated carbon under vacuum at 190±5°C for 20 min at 6 mm Hg. The bleached oil was then obtained by filtration under vacuum for further use. Enzymatic esterification in laboratory scale and pilot plant: For laboratory scale esterification, degummed, de waxed and bleached RBO was placed into a 250 mL conical flask fitted with standard B 24 joint. A predetermined amount of glycerol (either 40 % and / or 100 % stoichiometric excess) was added to the oil. 5% (w/w of oil) immobilised lipase was added. The oil was slowly heated to 500 C (at 4 mm Hg) and stirred with magnetic stirrer for 8 h. At regular 2 h interval aliquot was taken out and FFA was measured. For recycling purpose, esterification process has been carried out for 6 hours with Novozyme 435 as catalyst for sample II. In pilot plant, the bio esterification process was conducted in a 10 kg batch bioreactor with 4 kg of the high FFA degummed, de waxed and bleached oil. The enzyme (Novozyme 435) was recycled 10 times maintaining temperature 500 C at 4 mm Hg for six hours. Deodorization was carried out by conventional steam stripping at 180°C at 4 mm Hg. Quantitative determination of MG, DG and TG: The MG, DG, and TG content of crude and bio refined RBO were estimated by preparative thin-layer chromatography (PTLC) method. 0.5 mm thick layer of silica gel G ( 110-120 mesh) was applied to a 20×20 cm glass plate using 14 gm silica gel G and 28 mL distilled water. Plate was activated by heating at 110°C for 60 min. 0.1 gm exactly weighed oil was applied to the plate using a capillary and the plate was developed in 100mL hexane/ diethyl ether (80:20 vol/vol). Bands corresponding to MG, DG and TG were detected by iodine absorption and by Rf values [10] specific for each component. Each of the bands was scrapped from plate and extracted with chloroform. Each fraction was gravimetrically quantified as weight percentage of oil by evaporating chloroform under 4 mm Hg vacuum at 90°C. Statistical analysis of data: All experiments were completed in triplicate unless stated otherwise and the results are presented as mean ± standard deviation. Statistical differences of mean values were analyzed using student’s t-test in Statistica software.

CONCLUSION:

Deacidification and recycling of enzyme for high FFA RBO in laboratory scale and in bioreactor can be effectively utilized to produce quality oils. Nonspecific lipase Novozyme 435 is utilized for this esterification process which produces no by product, so isolation of used enzyme is rather simple. Recycling of enzyme can be successfully done in our study and also be implemented in industrial scale. The enzyme can be recycled sixty times or more which ultimately reduces the process cost. Another advantage of using enzyme for deacidifying purpose is that amount of antioxidant like oryzanol in the refined oil is nearly same as that of crude oil. It is quite beneficial for human health. Therefore, enzymatic deacidification process can be adopted in bench scale and is advantageous in deacidifying high FFA RBO compared to chemical and physical refining. The outcomeof our research work will also facilitate future researchers in better perceptive for the bioprocess to obtain quality product by using and reusing of enzymes in different chemical and biochemical sector