An efficient process for valorization of crude rice BRAN LYSO-gums

Abstract

Rice bran lyso-gums is a new class of by-product obtained during degumming of crude rice bran oil using bio-catalyst. Presently, this particular by-product is having very limited applications and is fetching negligible value to the industry. On the other hand, it contains lyso -phospholipids which has known applications in food, surfactant, pharmaceutical and cosmetics industries. The present invention relates to processes for possible value addition to crude rice bran lyso-gums- an unexplored by-product of rice bran oil refinery. It describes efficient processes for possible upgradation, modification, enrichment and purification of crude rice bran lyso-gums to obtain lyso-PC enriched rice bran lyso-lecithin having lighter colour by using various unit operation like drying, bleaching, solvent fractionation and chromatographic separation techniques in sequential manner.

Description

[0001] AN EFFICIENT PROCESS FOR VALORIZATION OF CRUDE RICE BRAN LYSO- GUMS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to an efficient process for possible value addition of crude rice bran lyso-gums [RBLG], an unexplored by-product of rice bran oil refinery. Particularly, the present invention relates to an efficient process to obtain Rice bran lyso-lecithin [RBLL] from crude rice bran lyso-gums generated during degumming of crude rice bran oil using various bio-catalysts. The present invention further relates to enrichment of lyso-Phosphatidyl choline [Lyso-PC or LPC] and Phosphatidylcholine [PC] to about 80% in the lyso-lecithin thus obtained.

[0004] BACKGROUND OF THE INVENTION

[0005] In recent years there has been a paradigm shift in the rice bran oil refining and processing industry. With various technological advances, most industries have shifted from conventional refining procedures to newer technologies.

[0006] Most industries are now using biocatalysts singly or in a mixture, for degumming crude rice bran oil. This, in turn, has led to the generation of gums that are different from the gums generated previously by water and acid treatment, and also from gums generated from any other crude oil. This is majorly lyso-gums, chemically different from conventional gums.

[0007] With growth in the edible oil processing sector, huge quantities of this new kind of gum - lyso- gums, are being generated in huge quantities. However, being new to its class, these lyso-gums hardly find their use in any application. The crude nature also hinders its usage in any high-end application. Industries presently are struggling to dispose of them. The lyso-gums therefore are presently being sold at a negligible price.

[0008] Value addition is the need of the hour for such kind of product. The rice bran oil refineries are struggling to dispose of this new kind of waste generated. However, industrialists are also looking for technological ways to modify this low value product, so as to utilize this in allied industries. This will not only make disposal of this waste easier but also help in generating revenue from this waste. The crude lyso-gums obtained hereby can be a potential source of lyso-Phosphatidyl choline (LPC) and Phosphatidylcholine (PC). These classes of molecules have huge importance in the cosmetic and pharmaceutical industries. These are used abundantly in various cosmetic and pharmaceutical industries as emulsifiers and nutraceuticals depending on their purity.

[0009] References may be made to Journal “J. Lipid Sci. Technol. 2008a, 40, 10-15”, wherein attempts were made to identify the various classes of lyso-phospholipids from rice bran gums. However, no attempts are reported to enrich LPC from rice bran lyso-gums.

[0010] References may be made to Journal “J Am Oil Chem Soc 92, 287-293 (2015)”, which describes the enrichment of LPC from canola lyso-gums only upto 15.8%. However, the present invention aims to develop process for enrichment of lyso-Phosphatidyl choline [Lyso-PC or LPC] and Phosphatidylcholine [PC] to about 80% in the lyso-lecithin thus obtained.

[0011] OBJECTIVE OF THE INVENTION

[0012] Main object of the present invention is to develop an efficient process for possible value addition to crude rice bran lyso-gums [RBLG].

[0013] Another object of the present invention is to obtain Rice bran lyso-lecithin [RBLL] from crude rice bran lyso-gums [RBLG] generated during degumming of crude rice bran oil using various bio-catalysts.

[0014] Yet another object of the present invention is to develop an efficient process for the preparation of lyso-lecithin with lighter colour by removing the dark coloured pigments and thereby reducing the overall colour of the product.

[0015] Yet another object of the present invention is to develop an efficient process for the preparation of lyso-lecithin fraction rich in lyso-phosphatidyl choline [lyso-PC] and phosphatidyl choline [PC] by selectively enriching the said fractions using appropriate solvent systems.

[0016] Yet another object of the present invention is to develop an efficient process for the preparation of bleached lyso-lecithin enriched with lyso-phosphatidyl choline [lyso-PC].

[0017] Yet another object of the invention is to develop a process for obtaining lyso-lecithin fraction rich in lyso-phosphatidyl choline [lyso-PC] and phosphatidyl choline [PC] by selectively removing other classes of phospholipids present like phosphatidyl ethanolamine [PE], phosphatidyl inositol etc. using chromatographic techniques. Yet another object of the present invention is to provide an efficient process for enrichment of the lyso-phosphatidyl choline [lyso-PC] and phosphatidyl choline [PC] to about 80% in the lyso- lecithin.

[0018] Yet another object of the present invention is to provide an efficient process to enrich the lyso- phosphatidyl choline [lyso-PC] and phosphatidyl choline [PC] present in the lyso-gums, so that the obtained gums can be used in cosmetic and pharmaceutical applications.

[0019] SUMMARY OF THE INVENTION

[0020] Accordingly, present invention provides a process for the preparation of lyso- phosphatidylcholine [Lyso-PC] and phosphatidylcholine [PC] enriched rice bran lyso-lecithin [RBLL] from crude rice bran lyso-gum [RBLG] comprising the steps of: i. providing crude rice bran lyso-gum [RBLG] obtained during industrial degumming of crude rice bran oil using bio-catalysts; ii. drying the RBLG as provided in step (i) in air oven at temperature in the range of 100- 105°C for a period in the range of 50 to 60 minutes to obtain dried crude RBLG; iii. mixing dried crude RBLG as obtained in step (ii) with a solvent followed by filtration to obtain dried purer grade crude RBLG; iv. solvent fractionating the dried purer grade crude RBLG as obtained in step (iii) using a solvent to obtain crude rice bran lyso-lecithin [RBLL]; v. bleaching the crude rice bran lyso-lecithin [RBLL] as obtained in step (iv) with bleaching agent to obtain bleached RBLL; vi. solvent fractionating the bleached RBLL as obtained in step (v) using a solvent to obtain Lyso-PC enriched RBLL; vii. bleaching the Lyso-PC enriched RBLL as obtained in step (vi) with bleaching agent to obtain bleached Lyso-PC enriched RBLL; viii. column chromatography of the Lyso-PC enriched RBLL as obtained in step (vii) using a solvent to obtain highly Lyso-PC and PC enriched RBLL; ix. column chromatography of the crude rice bran lyso-lecithin [RBLL] as obtained in step (iv) to obtain maximised Lyso-PC and PC enriched RBLL.

[0021] In an embodiment of the present invention, solvent used is selected from the group consisting of hexane, heptane, acetone, ethyl-acetate either alone or a mixture thereof.

[0022] In another embodiment of the present invention, RBLG and solvent ratio used in step (iii) is ranging between 1:1 to 1: 10.

[0023] In yet another embodiment of the present invention, the solvent used in step (iv) is selected from the group consisting of hexane, chloroform-hexane mixture, chloroform, acetone-hexane mixture, acetone, ethyl acetate-acetone either alone or a mixture thereof.

[0024] In yet another embodiment of the present invention, the ratio of dried purer grade RBLG and solvent used in step (iv) is ranging between 1:3 to 1:9.

[0025] In yet another embodiment of the present invention, the bleaching agent used in step (v) and (vii) is selected from hydrogen peroxide (H2O2), benzoyl peroxide (C14H1OO4), sodium chlorite (NaC102) either alone or a mixture thereof.

[0026] In yet another embodiment of the present invention, the solvent used in step (vi) is selected from the group consisting methanol, ethanol, iso-propanol, butanol, water either alone or a mixture thereof. In yet another embodiment of the present invention, the ratio of dried and bleached RBLL and solvent used in step (vi) is ranging between 1:2 to 1: 10.

[0027] In yet another embodiment of the present invention, the process as claimed in step (viii and ix) of claim 1, wherein solvent used is selected from the group consisting chloroform, acetone, methanol, ethanol, isopropanol either alone or a mixture thereof.

[0028] In yet another embodiment of the present invention, the process for the preparation of lyso- phosphatidylcholine [Lyso-PC] and phosphatidylcholine [PC] enriched rice bran lyso-lecithin [RBLL] containing about 50-55% Lyso-PC+ PC as in step (vi); 60-70% Lyso-PC+PC as in step (viii); and approximately 75-85% Lyso-PC+ PC as in step (ix).

[0029] DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention provides an efficient process for the utilization of a previously completely neglected by-product crude rice bran lyso-gums [RBLG] generated during degumming of crude rice bran oil using various bio-catalysts.

[0031] The present invention provides an efficient method for the preparation of lyso-lecithin from crude rice bran lyso-gums [RBLG] and the products thereof. The invention provides a simple, effective, and efficient process for isolation of the same.

[0032] Crude rice bran oil contains a high amount of waxes, more colour, and other impurities. It is therefore very tough to process the crude rice bran lyso-gums [RBLG] obtained after degumming of crude rice bran oil using bio-catalysts. The crude rice bran lyso-gums [RBLG] thus obtained was therefore subjected to bleaching in order to reduce its very dark colour.

[0033] The present invention provides a process for the development of bleached lyso-lecithin using various bleaching agents. The invention also provides a method for the development of bleached lyso-PC enriched lyso- lecithin from crude lyso-gums such the colour of the lyso-PC enriched lyso-lecithin is appreciably lighter.

[0034] The present invention provides processes for obtaining bleached lyso-phosphatidylcholine (LPC) and phosphatidylcholine (PC) enriched lyso-lecithin from crude rice bran lyso-gums [RBLG] obtained after industrial degumming of crude rice bran oil using bio-catalysts. The initial bleaching of rice bran lyso-lecithin showed the colour in the range of 12-14 Gardner scale unit.

[0035] The final bleached lyso-phosphatidylcholine(LPC) + phosphatidylcholine (PC) enriched lyso- lecithin showed the colour in the range of 8-10 Gardner scale unit.

[0036] The present invention provides a process for obtaining bleached lyso-phosphatidylcholine (LPC) and phosphatidylcholine (PC) enriched lyso-lecithin from crude rice bran lyso-gums, wherein more than 50% enrichment of lyso-phosphatidylcholine (LPC) + phosphatidylcholine (PC) was carried out in rice bran lyso-lecithin using the method of solvent fractionation.

[0037] The present invention provides a method for the development of further enriched Lyso-PC (nearly 60%) with very minute amount of other phospholipids and oil from lyso-PC enriched lyso- lecithin using the combined methods of solvent fractionation and column chromatography.

[0038] The present invention further provides a method for the development of isolating maximized Lyso-PC - enriched Rice bran lyso-lecithin [RBLL] with very minute amount of other phospholipids such that the Lyso-PC content was nearly 70%, while the total Lyso-PC + PC content in this fraction was approximately 80%.

[0039] The present invention further provides a process for obtaining bleached lyso-phosphatidylcholine (LPC) and phosphatidylcholine (PC) enriched lyso-lecithin from crude rice bran lyso-gums obtained after industrial degumming of crude rice bran oil using bio-catalysts, wherein rice bran lyso-phospholipid having a total LPC and PC content of approximately 80% (4 times more than the starting raw material) was obtained by using the method of column chromatography. The lyso-lecithin obtained is having a moisture content of less than 0.5%. The presence of impurities in the rice bran lyso-lecithin was also reduced to 0.5-1%.

[0040] The process for obtaining various grades to LPC enriched RBLL from crude rice bran lyso-gums [RBLG], an unexplored by-product of rice bran oil refinery, comprising of the following steps: i. Crude rice bran lyso-gum (RBLG) was obtained from Venkatrama Industries, Hussenpuram, Samalkot, East Godavari - 533440, Andhra Pradesh. The gum was obtained after the degumming of crude rice bran oil using bio-catalyst in a commercial rice bran oil refining plant. This lyso-gum [moisture content 1.2%] was taken, dried thoroughly in air oven at 100- 105 °C for 50 to 60 minutes such that the moisture content of the lyso-gums [dried crude RBLG] remains well below 0.5%. The removal of moisture is extremely important as the presence of this moisture leads to hydrolytic degradation of the lyso-gums, when it is subjected to temperature beyond room temperature (25-30°C). This entrapped moisture also leads to colour fixation in subsequent processing steps. ii. This crude dried RBLG was found to have impurity and foreign particles of nearly 6%. Various solvents like hexane, heptane, acetone, ethyl-acetate, a mixture of heptane- acetone and / or a mixture of hexane-acetone were added to it and thereafter subjected to filtration, via various membrane filters for obtaining RBLG free of most impurities. The solid-to-solvent ratio was varied from 1: 1 to 1: 10 in each case. The best ratio was fixed and it was passed through membranes of varying size for obtaining purer grade RBLG. The filtrate was centrifuged again at 8000g to 12000g to remove out any remaining impurities like bran fines. The supernatant was collected and was subjected to filtration where filters / membranes having pore size from 2pm to 0.2pm were used in order to remove the remaining impurities like solid impurities and mucilages. The filtrate obtained herewith was subjected to complete removal of the solvent first by rotary evaporator followed by solvent removal under high vacuum. The temperature of the water bath was maintained to a certain temperature so that it doesn’t degrade the gums. The insoluble impurity content came down to nearly 0.5- 1% from about 5-6%. iii. The dried purer grade RBLG, thus obtained, was subjected to solvent fractionation for removal of oil, waxes, colour pigments and others. Various solvents like hexane, chloroform-hexane mixture, chloroform, acetone-hexane mixture, acetone, ethyl acetate-acetone mixture were used. The solid-to-solvent ratio was varied from 1:3 to 1:9 in each case. The sequence of solvent was such that the polarity of the solvent mixture was increased in a gradient pattern. This allowed in efficient removal of the non-polar and slightly polar impurities. The best ratio was fixed and it was allowed to rest at temperature ranging from 4 to (-)10°C for a time period of 2-3hrs. After allowing it till the optimized time interval, the mixture obtained was subjected to centrifugation. The centrifugation speed was varied from 2000g to 10,000g for a time of 5-20 min. The supernatant contained majorly oil, waxes and other non-polar compounds and therefore was discarded. The whole process was repeated 3 times. Next, the insoluble fraction was collected and analysed for various components present in it and found to have enriched lyso-phospholipid (LPL) content. This is the crude rice bran lyso-lecithin (RBLL). iv. The crude rice bran lyso-lecithin, thus obtained, was collected such that the oil and wax content was nearly 30-35%, glycolipid content was 15-17% and total phospholipid and lyso-phospholipid content was nearly 45-50%. This RBLL obtained was very dark in colour. When measured using Gardner scale, the colour of this RBLL was seen to be nearly 18+. The colour of this RBLL was reduced using various oxidative bleaching agents. For this the RBLL were dissolved in hexane (w / v). The solid-to-solvent ratio was varied from 1:2 to 1: 10. The best ratio was fixed. Various oxidative bleaching agents like hydrogen peroxide (H2O2), benzoyl peroxide (C14H1OO4) and sodium chlorite (NaC102) were used either singly or in combination. The reaction was carried out under refluxing condition using an air condenser for nearly 6 hrs. The reaction mixture was dried by using rotary evaporator and finally under reduced pressure. The bleached RBLL obtained was checked for colour reduction using a Gardner colour scale. v. This dried RBLL obtained was used to prepare lyso-PC enriched RBLL. For this various solvent like methanol, ethanol, iso-propanol, butanol, water, methanol-water mixture, ethanol-water, isopropanol-water, methanol-ethanol mixture, and methanolisopropanol mixture were used. The RBLL was subjected to solvent fractionation using various solvent system as mentioned. The solid-solvent ratio was varied form 1:2 to 1: 10. The best ratio was fixed. The solvent concentration was varied from 80- 100% in each case. The time of reaction was varied from 30 min to 150 min. The optimum time of reaction was fixed based on relative rate of Lyso-PC enrichment. The LPC was enriched from 22% to 45%. Even PC was increased to 14%. This is the 1streport of enrichment of LPC to nearly 45% concentration and total LPC+ PC content of about 55% concentration. vi. The Lyso-PC - enriched RBLL thus obtained was subjected to bleaching to reduce its colour. As earlier, sodium chlorite (NaCICh) was used. The bleached Lyso-PC - enriched RBLL obtained was checked for colour reduction using a Gardner colour scale. It was seen that the colour was reduced from 18+ colour to 10+. vii. Present invention provides a process for further enrichment of Lyso-PC with very minute amount of other phospholipids and oil. For this, LPC-enriched RBLL prepared was taken and subjected to column chromatographic technique for isolating highly LPC - enriched RBLL. Silica gel having 60-120 mesh size was used as adsorbent while solvents with varying polarity like hexane, hexane-ethyl acetate mixture, chloroform, and hexane-chloroform mixture were used to elute out the oil and waxes present. The oil and wax free RBLL sample was eluted out from the column and it was subjected to complete solvent removal. Various solvents like chloroform, acetone, methanol, ethanol, isopropanol, mixture of chloroform methanol, mixture of chloroform-ethanol, mixture of chloroform - isopropanol, mixture of chloroformacetone, mixture of chloroform - acetone methanol chloroform - acetone- ethanol were used to elute out various factions of phosphatidyl ethanolamine (PE), phosphatidyl choline (PC) and finally lyso-phosphatidylcholine (LPC). The final LPC enriched fraction had LPC content of nearly 60%. An increase of nearly 15% LPC was seen from the LPC-enriched RBLL, which was used as the starting material for column chromatography in this case. viii. RBLL as prepared in step (iii) was taken as a starting material with an aim to obtain maximum enrichment of LPC in RBLL using column chromatographic technique. After eluting out the oil and waxes, the rice bran lyso-phospholipid fraction was then directly subjected to column chromatographic separation for isolating RBLL with maximum LPC. For this a mixture of 1 : 1 ratio of 60-120: 100-200 mesh silica column was used. The lyso- phospholipid fraction was loaded and remaining oil and waxes were eluted out using a mixture of hexane and ethyl acetate with a ratio varying from 1: 1 to 1:5. Various solvents like chloroform, acetone, methanol, ethanol, isopropanol, mixture of chloroform methanol, mixture of chloroform-ethanol, mixture of chloroform-isopropanol, mixture of chloroform- acetone, mixture of chloroformacetone methanol chloroform-acetone- ethanol were used to elute out various factions of lyso-phospholipid. At first phosphatidyl ethanolamine (PE) is eluted out, followed by phosphatidyl choline (PC) and finally lyso-phosphatidylcholine (LPC). Judicious increase of polarity was used throughout to avoid mixing of phosphatidyl inositol (PI) with LPC. The final LPC enriched fraction had LPC content of nearly 70%. The total LPC +PC content in this fraction was approximately 80%, which is almost 4 times more than the starting RBLL raw material.

[0041] From the present invention it is evident that simple unit operation like filtration, solvent fractionation could purify the lyso-gums obtained after degumming of crude rice bran gum using bio-catalysts. The lyso-lecithin was obtained when the lyso-gums was purified and all the oil, wax and other impurities like mucilages, bran fines, foreign particles were removed. The lyso- PC content in the lyso-lecithin was increased to more than 50% from the initial 20%. Further enrichment of the LPC content was possible by the use of chromatographic technique like column chromatography. By selective dissolution of lyso-PC using various solvent, lyso-PC enriched lyso-lecithin was obtained which had a lyso-PC + PC content of nearly 80%.

[0042] The surface tension of various grades of LPC - enriched RBLL lies in the range of 30-34 mN / m at 27°C. RBLL, various LPC-enriched RBLL, bleached RBLL will therefore find use in various surfactant and cosmetics industry. This process development for obtaining lyso-lecithin, lyso-PC enriched lyso- lecithin will help add value to this new class of un-utilized industrial by-product and thereby help in the overall sustainability of the rice bran oil refinery.

[0043] EXAMPLES

[0044] The following examples are given as a way of illustration only and should not be construed to limit the scope of the present invention.

[0045] EXAMPLE 1

[0046] 500g of crude rice bran lyso-gum (RBLG) was taken and the moisture content was found to be 1.2%. This gum was taken in large petri dish and thoroughly dried in an air oven at 100-105°C for Ihr. The weight of the lyso-gum was taken and the process was repeated until a stable weight was obtained. It was noted that the moisture of the lyso-gums came down to 0.45%. Moisture content below 0.5% is acceptable as this will prevent degradation of the lyso-gum in subsequent steps. The drying was carried out for 5kg scale and moisture content of 0.37% was observed in the final product.

[0047] EXAMPLE 2

[0048] 4 kg of the crude dried RBLG as mentioned in example 1 was taken and checked for the presence of impurities. It was found to contain nearly 6% of impurity. For this solvent was added to solubilize the gums and oil and then filtered to remove the insoluble impurities.

[0049] Step I

[0050] 50g of the dried RBLG was taken and various solvents such as hexane, heptane, acetone and ethyl acetate were added separately in 1: 1 ratio. The solubility of the RBLG in the solvents was found to be poor in all cases. Subsequently RBLG to solvent ratio was increased and it was found that optimum solubility was obtained at 1:9 solid to solvent ratio for selective solvents. It was followed by filtration. Maximum removal of impurity was noticed in case of hexane and ethyl acetate.

[0051] Step II 25g of the dried RBLG was taken and 1:9 ratio of hexane, hexane-ethyl acetate (80:20), hexaneethyl acetate (70:30), hexane-ethyl acetate (60:40), hexane-ethyl acetate (50:50), and ethyl acetate was added. These mixtures were passed through membranes of varying size for obtaining purer grade RBLG. The filtrate was centrifuged again at 10000g for 20min to remove out any remaining impurities like bran fines. The supernatant was subjected to filtration where filters / membranes having pore size from 1 pm was used in order to remove the remaining impurities like solid impurities and mucilages. The impurity content in the RBLG thus obtained was checked for each case. It was found that the RBLG treated with hexane- ethyl acetate (70:30) had the least impurity. The insoluble impurity content came down to nearly 0.5- 1% from about 5-6% for this case. For others, the impurity content was about 1.2-2%. The mixture was subjected to complete removal of the solvent first by rotary evaporator followed by solvent removal under high vacuum. The temperature of the water bath was maintained to a certain temperature so that it doesn’t degrade the gums.

[0052] Step III

[0053] 3kg of the dried RBLG was taken and subjected to removal of impurity using hexane-ethyl acetate (70:30) in the solid to solvent ratio of 1:9 as explained in step II of example 2. The final RBLG obtained was found to have presence of impurity of about 0.6%.

[0054] EXAMPLE 3

[0055] Step 1

[0056] 25g of the dried RBLG obtained in step III of example 2 was taken and subjected to solvent fractionation for removal of oils, fatty acids, waxes, glycolipids etc. For this, various solvents namely solvents like hexane, chloroform-hexane mixture, chloroform, acetone-hexane mixture, acetone, ethyl acetate-acetone were added in the ratio 1:3. Solubility of the RBLG in each case was checked. The amount of solvent was increased till optimized solubility was attained. 1 :5 ratio was found to be best suitable for solubilizing the RBLG. Non-polar solvents led to maximum dissolution of the RBLG hindering the selective removal of oils, waxes etc.

[0057] Step II 50g of the dried RBLG obtained in step III of example 2 was taken and 250ml of acetone -hexane mixture (70:30) was added. It was allowed to rest at temperature ranging from 4 to (-)10°C for a time period of 2hrs.The mixture obtained was subjected to centrifugation. The centrifugation speed was varied from 2000g to 5000g for a time of 5-15 min. The supernatant was collected and discarded as it contained the oils, waxes and other non-polar fractions. The weight of the dried insoluble fraction was found to be nearly 39g.

[0058] Step III

[0059] From the dried insoluble fraction obtained in step II of example 3, 25g was taken and subjected to acetone (100%) fractionation. The solid to solvent ratio needed to be increased further for efficient mixing of the solid fraction. Here the solid to solvent ratio was optimized to be 1 :9. After efficient mixing it was allowed to rest at temperature ranging from 4 to (-)10°C for a time period of 3hr for efficient removal of remaining oils, waxes, glycolipids etc. Next, the mixture was subjected to centrifugation at 10,000g for a time of 20 min. The supernatant was collected and discarded. The whole process was repeated 3 times. The weight of the insoluble and dried fraction was checked and it was nearly 21.2g.

[0060] Step IV

[0061] 3kg of the dried RBLG obtained in step III of example 2 was taken and subjected to solvent fractionation using the process optimized in step III of example 3. The insoluble fraction was collected and the weight was taken. The weight of the final product obtained was 2.20kg. It was analysed for various components present in it and found to have enriched LPL content. This product is termed as rice bran lyso-lecithin (RBLL).

[0062] EXAMPLE 4

[0063] Step I

[0064] 25g of the rice bran lyso-lecithin (RBLL) obtained in step IV of example 3 was collected and dissolved in hexane. The quantity of hexane was varied and was fixed at 1:6, RBLL: hexane. As this RBLL was very dark in colour having a colour of 18+ in Gardner scale, it was treated with various oxidative bleaching agents, namely: hydrogen peroxide (H2O2), benzoyl peroxide (C14H1OO4), 30% H2O2 (3%) and 50% H2O2 (3%). Also, 30% H2O2 (3%) + Benzoyl peroxide (1%) and 50% H2O2 (3%) + Benzoyl peroxide (1%) were tried. After carrying out the reaction under refluxing condition for 6 hrs, the colour was reduced to only 16+ when the bleaching agent used was 50% H2O2 (3%) + Benzoyl peroxide (1%). For the rest, colour was even darker.

[0065] Step II

[0066] Another bleaching agent - sodium chlorite (NaCICh) was tried. 2% and 4% sodium chlorite was added to the RBLL-hexane mixture as done in step I of example 4 and reaction was continued for 6 hr. Maximum colour reduction was seen when 4% sodium chlorite was used. The RBLL colour was then reduced to 14+ from 18+.

[0067] Step III

[0068] 3Kg of the rice bran lyso-lecithin (RBLL) obtained in step IV of example 3 was taken and subjected to bleaching using the process mentioned in step II of example 4. The colour of the RBLL was found to be 14+ Gardner scale.

[0069] EXAMPLE 5

[0070] Step I

[0071] 25g of the crude rice bran lyso-lecithin (RBLL) obtained in step III of example 4 was taken. This was subjected to alcohol fractionation to obtain lyso-PC enriched RBLL. For this, various solvents like methanol, ethanol, isopropanol, butanol, water, methanol-water mixture, ethanol- water, isopropanol-water, methanol-ethanol mixture, and methanol-isopropanol mixture were used. The solid-solvent ratio was varied form 1:2 to 1: 10. Depending on the solvent type, the solid: solvent ratio was fixed. Use of 100% alcohol did not give desirable enrichment. Therefore, water was introduced as a co-solvent along with the alcohols. The composition of the solvent was varied from 20:80-to 5:95 (water: alcohol). The time of reaction was varied from 30 min to 150 min. The optimum time of reaction was fixed based on relative rate of lyso-PC enrichment. In case of methanol-water, the yield of the soluble portion was nearly 50%. The LPC content was about 30%. For IPA-water the yield of the soluble fraction was about 35% with a LPC content of nearly 40%. The best results were obtained using ethanol- water as solvent in solvent to solid ratio of 1:5. The yield of the soluble fraction was about 41% with an LPC content of nearly 44% and PC content of 14%. Step II

[0072] 2kg of the crude rice bran lyso-lecithin (RBLL) obtained in step III of example 4 was taken and subjected to ethanol-water fractionation under optimized condition based on yield and LPC enrichment as in step I of example 5. The weight of the soluble fraction was found to be 853g, i.e. nearly 42.6% with an LPC content of nearly 43% and PC content of 14.2%. This is lyso-PC enriched RBLL, which has been used in the subsequent modifications.

[0073] EXAMPLE 6

[0074] Step I

[0075] 100g of the lyso-PC enriched RBLL obtained in step II of example 5 was taken and subjected to bleaching. Similar to step II of example 4, 4% sodium chlorite was added to the lyso-PC enriched RBLL-hexane mixture and reaction was continued for 6 hrs. After completion of the reaction, the solvent was removed completely using a rotary evaporator and then under high vacuum. The final colour of the bleached lyso-PC enriched RBLL was obtained as 10+ in Gardner scale. Thus, an appreciable colour reduction from initial 18+ to 10+ was attained.

[0076] EXAMPLE 7

[0077] Step I

[0078] 5g of the lyso-PC enriched RBLL obtained in step II of example 5 was taken and was subjected to column chromatographic technique for isolating highly LPC - enriched RBLL. For this silica gel having 60-200 mesh size was used to pack the column using chloroform as mobile phase. After adding the lyso-PC enriched RBLL slurry to the column, it was first subjected to removal of oil and wax. For this about 500-600ml of hexane was used initially to elute out maximum oil. Subsequently, lit of hexane-ethyl acetate (1:1) was used to elute out the remaining oil and waxes. Next, lit of chloroform was added to elute out any remaining traces of lipid, wax, fatty acid etc. Upon confirmation of complete removal of non-polar lipids, lyso-PC enriched RBLL sample, free from oil and wax, was eluted out from the column and it was subjected to complete solvent removal. The weight of the dried sample was nearly 3.5g.

[0079] Step II The product thus obtained in step I of example 7 was again subjected to column chromatography. The column was packed with silica gel having 60-200 mesh size using chloroform as mobile phase. Next, the glycolipid fraction was removed initially using 500ml chloroform followed by 500ml chloroform-acetone (1: 1) mixture. Lastly, acetone was used to elute out all traces of glycolipid completely. Next various grades of alcohol were used as the mobile phase to selectively elute out the various fraction of lyso-phospholipids to obtain even more enriched fraction of lyso-PC enriched rice bran lyso-lecithin. Chloroform-alcohol mixture was then used as mobile phase for selectively removing the various types of phospholipids. The percentage of alcohol was increased from 10% to 80%. Various alcohols like isopropanol, ethanol and methanol were used to elute out different fractions of phosphatidyl ethanolamine (PE), phosphatidyl choline (PC) and finally lyso-phosphatidylcholine (LPC). The final LPC enriched fraction had LPC content of nearly 60%. The weight of this LPC enriched RBLL fraction was 1.7gm giving a yield of nearly 34%.

[0080] EXAMPLE 8

[0081] Step I

[0082] In another variation of LPC- enrichment, 10g of the crude rice bran lyso-lecithin (RBLL) obtained in step IV of example 3 was taken and subjected to column chromatographic technique for isolating maximized LPC - enriched RBLL. For this, silica gel having 60-200 mesh size was used to pack the column using chloroform as mobile phase. After adding the RBLL slurry to the column, it was first subjected to removal of oil, wax and other non-polar lipid, lit of hexane was used initially to elute out maximum oil. Subsequently, lit of hexane-ethyl acetate (1: 1) was used to elute out the remaining oil and waxes. Next, 1.51t of chloroform was used to elute out any remaining traces of lipid, wax, fatty acid etc. It was followed by use of 500ml chloroform-acetone (90-10) to completely remove all waxes. This ensured complete removal of all non-polar lipid and initiated the removal of glycolipids. Next, 21t of acetone was run to elute out all the glycolipids present. The lyso-phospholipids remaining, were eluted out using methanol. The weight of this fraction was found to be 5.6gm.

[0083] Step II Slurry was prepared with 5g of lyso-phospholipid fraction obtained in step I of example 8 and loaded in a column with 1:2 of 100-200: 60-120 mesh silica using chloroform. This rice bran lyso-phospholipid fraction was then subjected to column chromatography for isolating LPC. The lyso- phospholipid fraction was loaded and still remaining traces of oil and waxes were eluted out using a mixture of hexane and ethyl acetate with a ratio varying from 1: 1 to 1:5 in isocratic pattern. Chloroform-alcohol solvent of various ratio was tried to elute out LPC. However, the products obtained did not have desired enrichment of LPC. Chloroform- acetone mixture was then used in varying ratio from 100:0 to 30:70. At first phosphatidyl ethanolamine (PE) is eluted out, followed by phosphatidyl choline (PC) and finally lyso-phosphatidylcholine (LPC). Judicious increase of polarity was used throughout to avoid mixing of phosphatidyl inositol (PI) with LPC. The final LPC enriched fraction had LPC content of nearly 70%. The weight of this fraction was 1.45gm. The total LPC +PC content in this fraction was approximately 80%, which is almost 4 times more than the starting RBLL raw material.

[0084] ADVANTAGES OF THE INVENTION

[0085] • This is one of the first reports of utilization of this rice bran oil refinery by-product- rice bran lyso-gum, which is presently having extremely low commercial value.

[0086] • Simple technological modification is required to obtain rice bran lyso-lecithin from the crude rice bran lyso-gum.

[0087] • The present work also uses unit operations to obtain various grades of improved and enriched rice bran lyso-lecithin.

[0088] • This particular modified and enriched lyso-lecithin is of special interest for its potential use in various industries particularly in surfactant, cosmetics and pharmaceutical industries. It has specific price advantage over egg-lyso-lecithin currently used for these purposes. Moreover, it can be projected as a vegan source of the specific type of surfactants.

Claims

We claim1. A process for preparation of lyso-phosphatidylcholine [Lyso-PC] and phosphatidylcholine [PC] enriched rice bran lyso-lecithin [RBLL] from crude rice bran lyso-gum [RBLG] comprising the steps of: i. providing crude rice bran lyso-gum [RBLG] obtained during industrial degumming of crude rice bran oil using bio-catalysts; ii. drying the RBLG as provided in step (i) in air oven at temperature in the range of 100- 105°C for a period in the range of 50 to 60 minutes to obtain dried crude RBLG; iii. mixing the dried crude RBLG as obtained in step (ii) with a solvent followed by filtration to obtain dried purer grade crude RBLG; iv. solvent fractionating the dried purer grade crude RBLG as obtained in step (iii) using a solvent to obtain crude rice bran lyso-lecithin [RBLL]; v. bleaching the crude rice bran lyso-lecithin [RBLL] as obtained in step (iv) with bleaching agent to obtain bleached RBLL; vi. solvent fractionating the bleached RBLL as obtained in step (v) using a solvent to obtain Lyso-PC enriched RBLL; vii. bleaching the Lyso-PC enriched RBLL as obtained in step (vi) with bleaching agent to obtain bleached Lyso-PC enriched RBLL; viii. column chromatography of the bleeched Lyso-PC enriched RBLL as obtained in step (vii) using a solvent to obtain highly Lyso-PC and PC enriched RBLL; ix. column chromatography of the crude rice bran lyso-lecithin [RBLL] as obtained in step (iv) to obtain maximised Lyso-PC and PC enriched RBLL.

2. The process as claimed in claim 1, wherein the solvent of step (ii) is selected from the group consisting of hexane, heptane, acetone, ethyl-acetate either alone or a mixture thereof.

3. The process as claimed in claim 1, wherein the dried crude RBLG and solvent ratio is ranging between 1: 1 to 1:10.

4. The process as claimed in claim 1 , wherein the solvent of step (iv) is selected from the group consisting of hexane, chloroform-hexane mixture, chloroform, acetone-hexane mixture, acetone, ethyl acetate-acetone either alone or a mixture thereof.

5. The process as claimed in claim 1, wherein the dried purer grade RBLG and solvent ratio is ranging between 1:3 to 1:9.

6. The process as claimed in claim 1 , wherein the bleaching agent is selected from hydrogen peroxide (H2O2), benzoyl peroxide (C14H1OO4), sodium chlorite (NaC102) either alone or a mixture thereof.

7. The process as claimed in claim 1, wherein the solvent of step (vi) is selected from the group consisting methanol, ethanol, iso-propanol, butanol, water either alone or a mixture thereof.

8. The process as claimed in claim 1, wherein the dried and bleached RBLL and solvent ratio is ranging between 1:2 to 1: 10.

9. The process as claimed in claim 1, wherein solvent of step (viii and ix) is selected from the group consisting chloroform, acetone, methanol, ethanol, isopropanol either alone or a mixture thereof.

10. The process for the preparation of lyso-phosphatidylcholine [Lyso-PC] and phosphatidylcholine [PC] enriched rice bran lyso-lecithin [RBLL] containing 60-70% Lyso-PC+PC as in step (viii) claim 1; and 75-85% Lyso-PC+ PC as in step (ix) claim 1.