Background of the invention Currently oils and fats are valued for their fatty acid composition having a bearing on health and diseases. They are also valued for imparting organoleptic properties such as aroma, flavour and mouth feel. Recent studies have created awareness on the minor constituents present in oils. These minor constituents, which are uniquely present in certain oils have been attributed with nutraceutical qualities and hence helpful in promoting health and prevention of diseases. The blending of appropriate oils correct the imbalance in the fatty acid composition of oils and also results in the enrichment of nutraceuticals derived from natural sources. However, blending or physical mixing of oils has some problems. Because of molecular size differences, two oils may be incompatible with one another and may form eutectic mixtures. Blending of semi-solid oils with liquid oils results in phase separation, which prevents uniform mixing of nutraceticals in blended oils and oils are not aesthetically acceptable. Rearrangements of fatty acids of triglycerol molecules of blended oils brought about by enzymatic interesterification alters the initial properties of oils and leads to the formation of new improved products for better quality. This also retains the nutraceuticals associated with the oils intact. Vegetable oils can be reacted together to yield a randomized composite oil having specific characteristics contributed from both oils. The present invention provides a process for the elimination of phase separation in vegetable oils and incorporation of nutraceuticals from natural sources. The resulted modified oil is clear, homogeneous with desired consumer appeal.

Various processing techniques have been studied for liquefying solid fats. The simplest process comprises merely mixing/blending of solid/semi solid oils with a liquid oil.

However, blending or physical mixing of oils has some problems. Because of molecular size differences, two oils may be incompatible with one another and may form eutectic mixtures.

Another known process comprises fractionating a solid fat to take a low-melting point fraction, but it will be apparent from, for example, oleins obtained by fractionation of palm oil that even the low-melting point fraction tends to be in solid form in seasons other than summer and thus the product obtained is only a semi-liquid oil. Further, these processes are

not so effective, since the amount of the solid fat which can be incorporated into the liquid oil is limited to an extremely small amount in order to obtain the intended oil. Blending of oils may not necessarily provide aesthetically acceptable oils. Besides, a combination of solid fat and liquid oil often results in phase separation, which prevents uniform mixing of nutraceuticals in blended oils. The oils in such blends behave as dependent entities. These processes are therefore not effective.

Under these circumstances, various processes based on transesterification reaction with lipase have been proposed. Lipase catalysed interesterification and transesterification reactions have been used to alter physico-chemical properties of vegetable oils. Such lipase catalysed reactions are also being used to target specific fatty acids at specific sites on the glycerol backbone. Biotechnological tools such as lipase catalysed interesterification reactions can be used to affect the rearrangement of fatty acid in triglyceride molecules of blended oils.

Reference is made to Ghosh, S. and Bhattacharyya, D. K. Journal of American Oil Chemists Society, Volume 74, Number 5, Page No. 589-592,1997, wherein the utilization of high-melting palm stearin by lipase catalysed interesterification reactions with sunflower oil, soybean oil and rice bran oil is reported. The resulting interesterified product has lower melting point and lower solid fat content. The carbon number of triglyceride compositions also changed significantly in the interesterified product. The authors'objective is not to eliminate solid phase in interesterified oil blend but to produce fatty material suitable for the production of trans-free shortening and margarine and therefore differs from this invention.

Reference is made to Foliga, T. A. , Petruso, K. and Feairheller S. H. , Journal of American Oil Chemists Society, Volume 70, Number 3, Page No. 281-285,1993, wherein, the interesterification of tallow and butterfat with high-oleic sunflower oil and soybean oil using 1,3-acyl-selective lipase or cis-9-C18-selective lipase allowed for either a decrease or increase in the solid fat content of the glyceride mixture. This work was carried out in an effort to provide interesterified blends of glycerides that have a wider range of thermal and melting properties than the starting fats and oils used. The drawback is not to eliminate the phase separation as described in the present invention Reference is made to Kurashige, J., Matsuzaki, N. and Takahashi, H. , Journal of American Oil Chemists Society, Volume 70, Number 9, Page No. 849-852,1993, wherein, the lipase catalysed reactions of palm oil blended with canola or soybean oil, the improvement in the fluidity of blended oils with canola oil than soybean oil was observed This work was carried out with an objective to improve the fluidity of palm oil to use as

stable and fluid frying oil. The drawback is not to eliminate two phases formed when liquid oil and solid fat were mixed.

Reference is made to Forssell, P. , Kervinen, R., Lappi, M., Linco, P. , Suortti, T. and Poutanen, K., Journal of American Oil Chemists Society, Volume 69, Number 2, Page No.

126-129,1992, wherein the reduction in the melting point of a tallow-rapeseed oil mixture was achieved by altering the triglyceride composition with enzymatic interesterification to 0 produce fat mixtures with improved melting properties. The drawback in this work is also not to eliminate the solid phase in the interesterified oil blend.

Reference is made to F. Takuma et al, Japan Patent No. 61293389, which discloses transesterification of palm oil and an odorless liquid starting oil with lipase taking advantage of 1,3-specificity. The drawback of this process is that the protocol used is not always satisfactory, since the amount of palm oil which can be incorporated is very low. Reference may also be made to Kokai, J. P. patent Nos. Sho 49-107304 and 64-81899, wherein a liquid oil is incorporated into an oil obtained from palm oil by fractionation and transesterification reaction is carried out with these oils. The drawback of this process is the obtained oil has an insufficient cooling resistance resulting in phase separation.

Reference is made to T. Nobuo, T. Koichi and T. Satoshi, US Patent 5,183, 675,1993, wherein a process for preparing salad oil by transesterifying a mixture of an oil or fat produced from palm oil and liquid edible oil is described. Drawbacks are although successful in producing liquid oil having a cooling resistance satisfying the standard salad oils, oil or fat produced from palm oil should have a defined physico-chemical property and the product has to be fractionated in the absence of solvent. This results in the partial or complete loss of 0- carotene, a valuable nutraceutical present in palm oil.

Objects of the invention The main object of the present invention is to provide a process for preparation of homogeneous blended oil which obviates the drawbacks as detailed above.

It is another object to provide a homogenous homogeneous oil with no phase separation by lipase catalysed interesterification reactions of blended oils enriched in specific nutraceuticals.

Another object is to provide an interesterification reaction to randomize fatty acids on triglycerides and obtain homogenous oils with desired consumer appeal.

Summary of the invention The present invention provides a process for preparation of homogenized blended oil which comprises blending of two or more vegetable oils having nutraceticals followed by

enzymatic interesterification with a lipase having 1,3 specificity in the absence of solvent which randomizes fatty acids on triglycerides resulting in the elimination of phase separation in blended oils. The interesterfied oils thus obtained are clear, homogeneous oil blends with desired consumer appeal.

Accordingly the present invention provides a process for the preparation of a homogeneous blended oil which comprises a) blending of two or more oils using mechanical stirrer under vacuum (0.1-1. 0 bar) for a period of 30 to 50 min; b) reacting the blended oil with an enzyme having 1,3 specificity in a ratio of oil to the enzyme 100 : 1 or 50: 1 for a period of 30 to 50 minutes in a temperature range of 37° C to 70 ° C to obtain a homogeneous oil.

In one embodiment, at least one of the component oils in the blended oil has a high saturated fatty acid content.

In another embodiment of the invention, the ratio of the high saturated fatty acid containing oil in the blended oil is 5 to 20% to liquid oil component.

In another embodiment, the oils are selected from vegetables and plants.

In another embodiment, the homogeneous liquid oil with no phase separation is obtained by homogeneously mixing semisolid oils selected from red palm oil and coconut oil with a liquid edible oil and subjecting the mixture to a 1,3-specific interesterification reaction between the fatty acid residue constituting the oil or fat and fatty acid residue constituting the liquid edible oil.

In another embodiment, homogeneous oil produced is enriched with specific nutraceuticals.

In yet another embodiment, weight ratio of semisolid oil to liquid edible oil is 20: 80.

In another embodiment of the invention, the two phases formed are separated by lipase catalysed interesterification reaction.

In another embodiment, semisolid oil is selected from group consisting of groundnut oil, mustard oil, coconut oil, soybean oil, sunflower oil, red palm oil and rice bran oil.

In yet another embodiment of the invention, liquid edible oil is selected from the group consisting of soybean oil, groundnut oil, mustard oil, rice bran oil, sesame oil and sunflower oil.

Brief description of the accompanying drawings Figure 1 is a flow sheet of the reaction of the invention.

Figure 2 represents lipase catalysed interesterification of blended oil containing red palm oil and soybean oil (20: 80). 1, Control blended oil having solid and liquid phases and 2, interesterified blended oil having single homogeneous liquid phase.

Detailed description of the invention The present invention provides a process for the preparation of a homogeneous blended oil composition using lipase catalysed interesterification reactions of blended oils containing solid/semi solid oil and liquid oil to eliminate the phase separation. Two or more vegetable oils were blended at appropriate ratios and the resulting blended oil was subjected to lipase catalysed interesterification reactions to eliminate phase formed between solid and liquid fractions of the blended oil.

The present invention provides a process for the elimination of two phases formed when palm oil is blended with liquid edible oil. In the process of the invention crude palm oil with varying physico-chemical properties can be used. The process also retains the i- carotene present in palm oil. Additionally the cloudiness of the oil was also significantly decreased after interesterification. Hence by interestrification process it is possible to overcome problem of phase separation, cloudiness of oil while retaining nutraceuticals intact.

It is to be understood that the present invention extends to oils treated by the present process. Throughout the present specification all percentages are by weight and the terms "oil"and"fat"are used interchangeably. By isothermal conditions"a temperature which is maintained substantially constant during interesterification reactions is meant. Slight fluctuations may occur without deviating from the present invention. The term"fatty acids" is taken to mean fatty acid moieties forming part of the triglycerides.

The immobilized lipase having 1,3-specificity usable in the present invention include, for example, Lipozyme (a product of Novo Nordisk A/S, Bagsvaard, Demark. ) which is produced by immobilizing a lipase having 1, 3-specificity derived from Mucor miehei by using an ion exchange resin as carrier with an activity of 278.84 Fmol free fatty acid min~lg~l expressed as peq of oleic acid released from olive oil per gram of immobilized lipase. The immobilized lipase usable in the present invention is not limited to Lipozyme but any lipase having 1,3-specificity which is carried on a known carrier is usable.

Oils for the purpose of blending were chosen from a number of commercially available oils which include groundnut oil, mustard oil, coconut oil, soybean oil, sunflower oil, red palm oil and rice bran oil. Physico-chemical characteristics of these oils are given in Table 1. Liquid edible oils usable as component (B) in the present invention include soybean oil, groundnut oil, mustard oil, rice bran oil, sesame oil and sunflower oil. They can be used

either singly or in the form of a mixture of two or more of them. Both oil components (A) and (B) used in the present invention may be crude, refined, bleached and deodorized oils.

The weight ratio of the oil or fat used as the component (A) to the liquid edible oil used as the component (B) in the present invention is 20: 80 in order to attain the object of the present invention which is to produce a clear homogeneous liquid oil having no phase separation and enriched with nutraceuticals. Clear homogeneous liquid oil can be produced in a satisfactory yield in a practical and economically advantageous manner by subjecting the blends of oils in the above-described weight ratio to the interesterification. The process can and preferably be performed in the absence of any solvent.

TABLE l : Physico-chemical characteristics and micronutrient content of oils used in the present investigation oil Peroxide Unsaponifiable Free fatty Micronutrients value matter (%) acid (3-carotene Oryzanol Tocopherols (meq/kg) value (%) (mg/100 g) (g/100 g) (mg/100 g) RPO 1. 86 2. 73 6.40 104.60 170.00 CNO 1.97 1. 66 0. 36 2. 70 RBO 1. 92 2. 25 0. 60 1. 30 160.00 SNO 1. 97 1. 82 0. 056 40. 00 SBO 3.97 1. 68 0. 13 125. 00 GNO 4. 01 1. 13 0. 05 26. 60 MO 5. 91 0. 95 0. 674 57. 60 RPO: Red palm oil, CNO: coconut oil, RBO: Rice bran oil, SNO: Sun flower oil, SBO: soybean oil, GNO: Groundnut oil, MO: mustard oil Interesterification reaction in the present invention is conducted at about 37°C to 60°C, particularly around 37°C. Interesterification reaction in the present invention is performed batch wise for period up to 72 hr. High temperature and longer reaction time are desirable for increasing reaction (interesterification reaction) velocity, but pose a problem of deactivation of the enzyme and, in addition, the amount of free fatty acids formed by the hydrolysis (side reaction) is increased, which will not satisfy the standard of edible oils (Prevention of Food Adulteration Act (1954, India).

The concentration of the immobilized lipase used in the present invention is 0.1 to 10% (w/w), preferably 1.0% (w/w) in order to attain the object of the present invention. As low as 0. 1%-immobilized lipase in the reaction was sufficient to remove the phase separation and the blended oil became a clear viscous solution. However upon storage the clear oil started becoming cloudy and ultimately phase separation was observed with time depending

upon the concentration of the lipase used in the reaction. With 0.1% to 0. 5% lipase the clear oil was observed up to 6 days followed by turbidity up to 8 days and by lOth day a clear phase separation in the blended oil was observed. However with 2.5%-10% lipase clear liquid oil was observed even after 30 days of storage. However with an increase in the amount of enzyme used for interesterification reactions, there was an increase in free fatty acid content of modified oils ranging from 2.34-9. 76% (control 0.99%). The modified oils with 1.0 % immobilized lipase for 1.0 hr are clear, homogenous and suitable oils with 3.04 % free fatty acids. Modified oils stored for a period of six months remained as clear oils while blends showed clear phase separations (Figure 2). When the ratio of the immobilised lipase to the oil is in the range of 1.0% to 2.0% (w/w), the clear oil can be produced in an economically more advantageous manner.

According to the present invention, blending and interesterification of appropriate oils correct the imbalance in the fatty acid composition of vegetable oils and also results in the enrichment of nutraceuticals derived from natural sources. The modified oil produced by the present invention by rearranging fatty acids in triglyceride molecules is clear with no phase separation. In addition, no significant change in the content of nutraceticals such as carotene, oryzanol and tocopherols was observed in the modified oil. A flow chart for the preparation of interesterified oil is given in Figure 1.

The following examples are given by way of illustration of the present invention and therefore should not be constructed to limit the scope of the present invention.

EXAMPLE-1 a) Preparation of blended oil: 100 grams of crude palm oil having ß-carotene content of 1046 ppm and other physicochemical properties listed in Table 1 was homogeneously mixed with 400 grams of sunflower oil in a flat bottom 1000 ml narrow mouthed flask at 40° C under vacuum and stirred with a magnetic stirrer for 45 minutes. The phase separation of this blended oil was monitored visually. A strong two phases, solid phase at the bottom and liquid phase on top was noticed within 24 hours at room temperature after blending. This two phase separated blended oil was used for lipase catalysed interesterification reaction. b) Preparation of homogenized blended oil by interesterification : 25 g of the above-described oil blend was subjected to interesterification reaction with 250 milli grams of immobilized lipase having 1, 3-specificity at 37° C for one hour with constant shaking (100 rpm) in a incubator shaker. After the one hour of incubation period the oil was decanted. The immobilized enzyme was washed with 30 ml of hexane to collect traces of oil. Hexane was removed by flash evaporation and then by keeping in vacuum oven at 71°C and 15 inch

vacuum. The modified oil was flushed with nitrogen and stored in dark at room temperature and visual observations were made for phase separation. Immediately after interesterification single-phase oil was obtained.

Control experiment was performed by same method described above except that immobilized lipase was not added to blended oil. Strong phase separation was noticed within 24 hours at room temperature. The oil obtained is not aesthetically acceptable and does not have desired consumer appeal. Therefore this process is economically unsatisfactory.

Table 2: Reverse phase HPLC analysis of tryglycerides in blended and interesterified oil of red palm oil and sunflower oil (20: 80).

Retention Relative Relative concentration of % change TG in time of concentration of TG TG in interesterified oil intersterified oil TG (min.) in blended. oil 5.47 Nil 1.21 New 5.73 0. 75 2. 18 190. 22 6.49 0. 40 1. 02 151. 11 6.99 0. 89 2. 70 203. 42 7.67 1. 10 2. 80 154. 02 8.49 0. 290. 316. 89 f 8.86 0. 13 0. 31 143. 42 9.41 0.57 0.42 25.94 # 10.33 9.02 7.68 14.81 # 11.63 23. 60 21. 15 10. 39 l 13.41 30. 4828. 506. 46 J. 15.87 27. 21 25.63 5.77 # 18.54 Nil 0.63 New 19.81 3. 84 3. 89 1. 07 23. 39 0. 55 0. 72 31. 04 1 TGs, triglycerids, T, increase in concentration of triglyceride and 1, decrease in concentration

of triglyceride No significant changes in the content of nutraceuticals such as ß-carotene and tocopherol and marginal increase in free fatty acid value was observed in the interesterified oil. Modified oils stored for a period of six months remained as clear oils, which is due to increase in the concentration of minor triglycerides in interesterified blended oil in the region of 5. 47-8.86 minutes and 18.54-23. 39 minutes retention time (RT) and appearance of new triglyceride species at RT of 18.54 min. Considerable amount of decrease in relative concentrations of triglycerides corresponding to RTs 10.33 and 11.63 were also observed in the interesterified oil compared to blended oil (Table 2).

EXAMPLE-2 (a) Preparation of blended oil: 100 grams of refined coconut oil having physicochemical properties listed in Table 1 was homogeneously mixed with 400 grams of rice bran oil by the same method as in Example 1. Within 24 hours at room temperature after blending a cloudiness and turbidity was observed in the blended oil. This turbid blended oil was used for lipase catalysed interesterification reaction.

(b) Preparation of homogenized blended oil by interesterification: 25 g of the above- described oil blend was subjected to interesterification reaction with 250 milligrams of immobilized lipase by the same method as in Example 1. Modified oil was flushed with nitrogen and stored in dark at room temperature and visual observations were made for phase separation and cloudiness. Immediately after interesterification single-phase oil was obtained.

. A control experiment was performed by same method as described above except that the immobilized lipase was not added to the blended oil. A cloudiness and phase separation was noticed within 24 hours at room temperature. The oil obtained is not aesthetically acceptable and does not have desired consumer appeal. Therefore this process is economically unsatisfactory.

Table: 3. Reverse phase HPLC analysis of tryglycerides in blended and interesterified oil of coconut oil and rice bran oil (20: 80).

Retention Relative concentration Relative concentration of % change of TG in <BR> <BR> time min. of TG in blended oil TG in interesterified oil intersterified oil<BR> time (min.) of TG in blended oil TG in interesterified oil intersterified oil 5. 65 Nil 1. 20 New 6. 09 0.07 0.84 1185. 32 T 6. 38 0.51 1.19 134. 87# 6. 73 2.35 3.15 33. 65 7.13 9.63 10.62 10. 22 7.64 13.20 13.99 5. 99 t 8. 22 15.01 15.76 6. 99 8. 99 12.71 13.06 2. 73 1 9. 95 10.91 10.53 3. 43 i 11.18 13.09 10.02 23. 42 12.72 13.05 10.64 18. 45 1 15. 34 8.48 8.04 5. 16 <BR> <BR> 17.89 0.99 0.95 4.46#<BR> <BR> <BR> <BR> <BR> <BR> TGs, triglycerids, 1, increase in cocentration of tnglycerlde and 2, decrease m concentration of triglyceride No significant changes in the content of nutraceuticals such as oryzanol and tocopherol and marginal increase in free fatty acid value was observed in the interesterified oil. Modified oils stored for a period of six months remained as clear oils. HPLC analysis of

triglycerides given in Table 3 indicate that new triglyceride species representing peak corresponding to retention time (RT) 5.65 was observed in interesterified oil. This peak is absent in blended oil. About 13 fold increase in the relative concentration of the triglyceride corresponding to RT 6.09 and significant increase in relative concentration of triglyceride corresponding to RT 6.38 was observed in interesterified oil compared to blended oil.

Considerable amount of decrease in relative concentrations of triglycerides corresponding to RTs 11.18 and 12.72 were observed in the interesterified oil compared to blended oil.

Therefore it is evident from the triglyceride analysis that lipase catalysed interesterification reaction rearranges the fatty acid residues on triglyceride molecules in oils. This randomization of fatty acids results in the elimination of phase formed between solid fat and liquid oil. Hence by interestrification process described in present invention, it is possible to overcome the problem of phase separation and cloudiness of oil while retaining the nutraceuticals intact.

EXAMPLE 3 a) Preparation of blended oil using red palm oil, soybean oil and coconut oil (20: 15: 65) and interesterification: 75 g of crude red palm oil, 100 g of soybean oil and 325 g of coconut oil were blended by the same method as in Example 1. This blended oil resulted in phase separation when stored at room temperature. This phase separated oil was subjected to interesterification reaction as in Example 1. This interesterification reaction yielded clear oil eliminating phase separation and cloudiness. A control experiment was performed by same method as described above except that the immobilized lipase was not added to the blended oil. A clear phase separation, cloudiness and turbidity was noticed within 24 hours at room temperature. The oil obtained is not aesthetically acceptable and does not have desired consumer appeal. Therefore this process is economically unsatisfactory. Triglyceride analysis of this interesterified oil by HPLC indicate that there is a substantial increase in the relative concentrations of the triglycerides corresponding to RTs of 6.20, 6. 49 and 6.85 compared to blended oil. Significant amount of decrease in relative concentrations of triglycerides corresponding to RTs 13.10, 13.31 and 18.18 were observed in the interesterified oil compared to blended oil (Table 4). Therefore it is evident from the triglyceride analysis that lipase catalysed interesterification reaction rearranges the fatty acid residues on triglyceride molecules in oils. This randomization of fatty acids results in the elimination of phase formed between solid fat and liquid oil. No significant changes in the content of nutraceticals such as oryzanol, (3-carotene, tocopherol and marginal increase in free fatty acid value was observed in interesterified oil.

Table: 4. Reverse phase HPLC analysis of tryglycerides in blended and interesterified oil of soybean oil, red palm oil and coconut oil (20: 15: 65).

Retention Relative concentration Relative concentration of % change of TG in <BR> <BR> time min. of TG in blended oil TG in interesterified oil intersterified oil<BR> time (min.) of TG in blendede oil TG in interesterified oil intersterified oil 6.20 0. 85 2.48 191. 40t 6.49 0.90 1.78 98. 89 6.85 2.26 3.23 42. 85 7.26 8.18 9.28 13. 50 7.77 11.30 12.28 8. 73 t 8.36 13.38 14.33 7. 14 9.14 12.80 13.36 4. 41 t 10.06 13.49 13.62 0. 9935 t 11.35 12. 79 10.61 17. 03 13.10 5.67 3.25 42. 63 13.31 5.08 3.55 30. 04 15.54 11.3654 10.5566 7. 111 <BR> <BR> 18.18 1.9519 1.6506 15.43 #<BR> <BR> <BR> <BR> <BR> <BR> <BR> TGs, triglycerids, #, increase in cocentration of triglyceride and #, decrease in concentration of triglyceride Blending of semisolid oils with liquid oils results in the phase separation. This prevents the uniform mixing of nutraceuticals present in vegetable oils and this phase separated oil is not aesthetically acceptable. It is evident from the triglyceride analysis by HPLC that lipase catalysed interesterification reaction rearranges the fatty acid residues on triglyceride molecules in oils. This randomization of fatty acids altered the initial properties of oils which resulted in the elimination of phase formed between solid fat and liquid oil.

Hence by interestrification process it is possible to overcome the problem of phase separation and cloudiness of oil while retaining the nutraceuticals intact.

The main advantages of the present invention are : 1. Use of immobilized lipase with 1,3-specificity enables interesterification reactions and elimination of phase separation and cloudiness in vegetable oils 2. Lipase catalysed interestrification reactions of selected oil blends provide appropriate fatty acids for modification and also incorporate nutraceuticals for value addition to modified oils. Such type of oils will be valued by the customers since the nutraceuticals . are added from natural sources.

3. Lipase catalysed interestrification reactions of blended oils change functionality of oils and fats by randomizing fatty acids in triglyceride molecule, leading to formation of new improved products of better quality.