Title of Invention

"A PROCESS FOR THE PREPARATION OF ORYZANOL FLAKES FROM THE ORYZANOL ENRICHED SAPONIFIABLE FRACTION OF RICE BRAN OIL"

Abstract

The present invention relates to a process for crystallization of oryzanol flakes from oryzanol enriched saponifiable fraction of rice bran oil (RBO) which can be obtained by leaching of saponified and dehydrated rice bran oil soapstock. Attempts in the direction for effective utilization of the byproduct of RBO refining (soapstock) for value addition will definitely improve the economics of RBO refining.

Full Text

A PROCESS FOR THE PREPARATION OF ORYZANOL FLAKES FROM THE ORYZANOL ENRICHED SAPONIFIABLE FRACTION OF RICE BRAN Oil Technical Field The present invention relates to a process for preparation of oryzanol flakes from oryzanol enriched saponifiable fraction of rice bran oil (RBO) which can be obtained by leaching of saponified and dehydrated rice bran oil soapstock. Background Art Now the utility of soapstock is to generate the soap for toiletries and detergent industries. This application does not fetch lucrative price for soapstock. Rice bran oil soapstock contains nutritionally important health promoting component like ory/anol. Hence, there is need to develop a simple cost competent separation processes for the-isolation of oryzanol from rice bran oil soapstock. Attempts in the direction for effective utilization of the byproduct of RBO refining (soapstock) for value addition will definitely improve the economics of RBO refining. Oryzanol is gaining importance in food, pharmaceutical and cosmetics due to its increasing applications. The beneficial and nutritional effects of oryzanol on human health are well discussed (Pikonen et al., 2000, Plant sterols: Biosynthesis, biological function and their importance to human nutrition. Journal of the science of food and Agriculture 80, 939-966). The expansive pharmaceutical application of oryzanol include hypocholesterolemic activity (Seetharamaiah and Chandrasekhara, 1989, Studies on hypocholesterolemic activity of rice bran oil, Artherosclerosis 78, 219-223). the-emerging application of oryzanol in cosmetic preparations includes it usefulness in treatment of skin related disorders like melanin related disorder (Tatsu et al., 1993. .JP 05225037) and minimizing the wrinkles in aged women (Tatsu et al., 1993, .IP 0530526) In food application, oryzanol proves its usefulness as an antioxidant (Minami and Morito. 1982. Conditions for using oryzanol and its utilization in food, New Food Industries 24(10) 49-53). All these beneficial effects of oryzanol on human health generated global interest in developing viable separation method for oryzanol from the rice bran oil soapstock. The attractive cost competent byproduct (soapstock) as starting material for value addition is major motivation here. Reference can be made to that of Masao and Yoshizane (1968) [Oryzanol Japanese patent, 68, 12730, 29 May, 1968, (chem. Abst., 1968, 69, 107775f| which refers to gas (CO2) antisolvent precipitation of oryzanol from alcoholic (ethanolic or methanolic) solution of alkaline oil cake of rice oil to give 85 % oryzanol. The separation principle utilized here is, oryzanol solubility can be reduced considerably by passing carbon dioxide (gas) at elevated temperature and pressure through alcoholic solution of alkaline rice cake of rice bran oil having oryzanol. The limitation of this process is that though this claim has a potential to perform on a large scale, failure may be expected to separate the interfering impurities especially when impurities are sufficiently insoluble in gas solvent mixture. Further, this process cannot account for the variation in the starting material (soapstock / unsaponifiable oryzanol enriched fraction). Another reference can be made to that of Masao and Yoshizane (1968) [Oryzanol Japanese patent, 68,12725, 29 May, 1968, [(chem. Abst., 1968, 69, 107774g)] which refers to a process for isolation of oryzanol from alkaline oil cake of rice oil The process comprises of deacidification of oil without solvent, followed by antisolvent precipitation of oryzanol in a solvent such as trichloethane or benzene or hexane or mixture of benzene and hexane (1:1) by employing water and carbon dioxide as antisolvents. The drawback of this method is that it utilizes either chlorinated solvent or aromatic solvent (benzene) which are neither safe nor food grade. Further, although this method has a potential to perform on a large scale however failure may be expected to separate the interfering impurities especially when impurities are sufficiently insoluble in gaseous solvent mixture like gums. Another reference can be made to that of Junichi et al., (1977) [Process for crystallizing oryzanol JP52007415A2 (01/20/1977)] where a simple speedy process for crystallization of oryzanol from oryzanol composition is claimed to provide 95 % pure oryzanol. The drawback of this method is that it refers to a model system and that too with limited impurities and it may not work for a system like soapstock or unsaponifiable fraction having wide range of impurities (real system). Another reference can be made to that of Yoshiaki and Isao (1978) [Purification of gamma-oryzanol JP 53130412] where a process for recrystallization of oryzanoi from crude oryzanol is claimed by employing specific solvent. Since it is recrystallization process, direct comparison can not be made with the present crystallization process. Another reference can be made to that of Nobuyoshi (1983) [Concentration of each constituent from crude or purified oryzanol .IP 58021619] where a solvent which does not dissolve oryzanol such as methanol or water was suggested to add to an organic solution of crude or purified oryzanol (in acetone) to achieve partial precipitation of oryzanol components. The limitation of the process is that it is precipitation process involving higher quantities of antisolvents leading to loss of selectivity. This is in contrast with the present process which is crystallization process involving lower quantities of antisolvents leading to higher selectivity. Another reference can be made to that of Kobayshi and Tokuo (1988) [Concentration and purification of constituent component of oryzanol JP 63104948] where concentration and purification of oryzanol is achieved by multistage recrystallization or multistage solid/liquid extraction (leaching) employing easily recoverable solvent or solvent mixture from an alcohol (methanol, propanol, butanol etc.) or hydrocarbon (hexane, cyclohcxanc, tolufenc etc.). The limitation of this process is that it involves a multistage process and hence can not be directly compared with the present single stage process. Another reference can be made to that of Kimura (1988) [Separation of 24-methylene cycloartanol ferulate JP 63014797] where a specific crystallization operation for separation of 24-methylene cycloartanol ferulate from crude or purified oryzanol was discussed by employing solvent mixture of organic acid ester, ketone and alcohol. The limitation of this process is that it pertains to only one component of the oryzanol mixture and that too achieving only 55 % purity. Another reference can be made to that of Seetharamaiah and Prabhakar (y-oryzanol content of Indian rice bran oil and its extraction from from soap stock. J. Food Sci. Technol. 23: 1986: pp 270-273) where a method for isolation of oryzanol was discussed which comprises of liquid/liquid extraction (organic/aqueous extraction), column chromatography (alumina), crystallization and recrystallization. The solvent used for crystallization and recrystallization was methanol and mixture of methanol &. acetone (2:1) respectively. The limitation of the process is that it is a laboratory process, which is not feasible to be translated to a production scale. Another reference can be made to that of Saska M. and Rossiter G.J. (1998) [Recovery of -oryzanol from rice bran oil with silica-based continuous chromatography, J. Am. Oil Chem. Soc. 75(10), 1421-1427] where recovery of oryzanol from degummed and dewaxed rice bran oil was discussed by using silica based continuous chromatography followed by crystallization in a solvent medium like heptane. The method is applicable to isolate the oryzanol from oil not from soapstock and hence direct comparison can not be made with the present process. Another reference can be made to that of Singh et al., (2000) [J. Oil Technol. Assoc. India, 32(2), 55-58]. The isolation of oryzanol from crude rice bran oil was achieved by using four steps namely: chromatography, rechromatography, purification, and crystallization. Here, the final crystallization was carried out in a-solvent mixture of methanol and acetone in the ratio 2:1. After crystallization white crystals of 98 % pure oryzanol was obtained. The method is applicable to isolate the oryzanol from crude oil and that too interfering impurities are removed prior to crystallization by chromatography and hence direct comparison can not be made with the present process. Another reference can be made that of Rao et al., (2002) [Process for the isolation of oryzanol from rice bran oil soap stock, US patent # 6,410,762] which refers to a process for the isolation of oryzanol from rice bran oil soapstock. This process comprises of saponification of neutral oil present in the soapstock, conversion of soapstock into anhydrous porous soapstock noodles and subsequent leaching followed by crystallization, column chromatography and recrystallization to yield 90 % oryzanol with overall 70 % recovery. The limitation of the present process is that it involves two steps before final crystallization namely crystallization and chromatography (which is difficult to scale up) to achieve the reported purity, where as the present process is a single step process and hence direct comparison can not be made. The oryzanol content of unsaponifiable fraction is found to be sensitive to the kind of soapstock used for leaching and the type of leaching process adopted. The leaching process adopted in this process takes care of it. Objects of the invention The main object of the present invention is to provide a simple process for crystallization of oryzanol from oryzanol enriched fraction. Another object of the present invention is to provide suitable solvent mixture for crystallization of oryzanol. Still another object of the present invention is to provide a method of fractional crystallization for partial separation of impurities. Yet another object of the present invention is to provide a method to obtain free flowing highly purified powder of oryzanol. Summary of the Invention Accordingly present invention provides a simple process for crystallization of oryzanol from oryzanol enriched fraction, which comprises (a) nonselective temperature assisted dissolution of oryzanol enriched unsaponifiable fraction in a suitable solvent mixture; (b) fractional precipitation of mucilaginous interfering impurities; and (c) crystallization of oryzanol from supernatant by either single method or combination of known methods to improve selectivity of crystallization. Detialed Description of the Invention Accordingly the present invention provides a process for the preparation of oryzanol Hakes from the oryzanol enriched saponifiable fraction of rice bran oil (RBO) soapstock wherein the oryzanol shows purity and yield as ranging between 60-65% and 70-75% respectively the said process comprising steps of: a) leaching the saponified and dehydrated rice bran oil soapstock to obtain oryzano! enriched unsapomfied fraction, (b) dissolving the unsaponified fraction of step (a) in a mixture of organic solvent selected from mixture of acetone and methanol in a ratio ranging between 1:3 tol:4 at a reflux temperature, (c) cooling the mixture of step (b) gradually to a temperature in the range of 25 to 30C allowed to stand at this temperature for a period of 30 to 120 minutes to precipitate mucilaginous impurities. d) separating the mucilaginous impurities of step (c) to obtain a clarified supernatant, e) crystallizing the supernatant of step (d) at a temperature range of 5 to 10°C over a period of 10 to 16 hours to obtain crystallized oryzanol, f) separating and desoivating the crystalline oryzanol of step (e) to obtain flakes of oryzanol, and g) micronizing the orazonal flakes of step (f) to obtain flavouring powder. In an embodiment of the invention, the solvent used in step (b) is a mixture of acetone and methanol. Still another embodiment, the ratio of solvent acetone: methanol used in step (b), ranges between 1:3 to 1:4. Yet another embodiment, the ratio of oryzanol enriched unsaponiifable fraction and organic solvent mixture is in the range of 1:3 to 1:6. Yet another embodiment, the crystallization in step (e), may be performed adapting methods selected from temperature controlled fractional crystallization, temperature controlled antisolvent crystallization, antisolvent crystallization, high pressure gas antisolvent crystallization or combinations thereof Yet another embodiment of the invention provides a process, wherein in step (f), desolvation is performed either by drying under vacuum or by distillation. In an embodiment of the present invention nonselective dissolution of oryzanol enriched unsaponifiable fraction of rice bran oil soapstock may be achieved by temperature assisted process in a suitable mixtures of organic solvents such as acetone and methanol (1: 3 to 4) at solid to solvent ratio of 1:5. In an another embodiment of the present invention, fractional precipitation of mucilaginous interfering impurities may be achieved by gradually lowering temperature to 22-29°C over a settling time period ranging from 45-90 minutes. In yet another embodiment of the present invention crystalline fraction may be desolventizated preferably by vacuum drying/distillation to give oryzanol flakes, which may be converted to free flowing powder by commination (size reduction). Characterization of starting material for crystallization was unsponifiable fraction, which was obtained by leaching of, pretreated, sponified and dehydrated soapstock. The unsaponifiable fraction is highly enriched in oryzanol. In the present case, this fraction was containing nearly 50% of oryzanol. The oryzanol content of unsaponifiable fraction is sensitive to the kind of soapstock used for leaching and type of leaching employed. This fraction neither contains any acidic nor basic residues. In the present invention following steps are involved in the process. (1) Oryzanol enriched unsaponifiable fraction of rice bran oil soapstock is mixed with mixtures of suitable organic solvents such as acetone and methanol (1: 3 to 4) at particular solid to solvent ratio of 1:5 and refluxed at elevated temperature to facilitate the complete solubilization of oryzanol throughout the solvent mixture. (2) Hot oryzanol enriched refluxed solvent mixture is subjected to fractional precipitation at predetermine temperature (22-29 °C) for predetermine settling time ranging from 45 to 90 minutes to strip solid mucilaginous interfering impurities. (3) The clarified oryzanol rich supernatant may be subjected to any of the method of crystallization or combination of them in order to improve selectivity of oryzanol isolation. (a) Temperature controlled fractional crystallization. (b) Antisolvent crystallization (c) Temperature controlled antisolvent crystallization. (d) High pressure gas antisolvent crystallization (4) The recovered crystalline fraction on desolventization preferably by vaccum drying/distillation gives oryzanol flakes, which can be converted to Micronized free flowing powder by comminution (size reduction). The separation principle utilizes first "non selective temperature assisting dissolution" of crude oryzanol in a solvent mixture of acetone and methanol (suitable polarity and methanol to acetone ratio of 3.8:1.2) and then availing the fractional precipitation of impurities from solution by employing temperature controlled precipitation for predetermine duration. The novelty in the separation lies in the selection and production of starting material for crystallization (which can be obtained by leaching of saponified and dehydrated soapstock in a single stage in most simple way) and separation of interfering critical impurities such as gums. Further, the process is versatile enough to accommodate the changes in processing conditions during crystallization brought by either single method or combination of several methods which provide scope to improve selectivity of oryzanol isolation. Process provides a method of producing free flowing powder of oryzanol for improved applications in foods and pharmaceuticals which is other wise difficult to comminute (size reduction) without crystallization due to presence of critical mucilaginous interfering impurities. The following examples are given by the way of illustration of the present invention and should not be construed to limit the scope of the present invention. Exam pie 1 50 gm of oryzanol enriched fraction (50 to 55 % oryzanol) unsaponifiable fraction [prepared as per Indira et al., (2002), patent #2] was mixed with suitable mixtures of organic solvents such as acetone and methanol (1.2: 3.8) at particular solid to solvent ratio of 1:5 and refluxed at elevated temperature of 80 °C for 1 hour to facilitate the nonselective dissolution. Then, the temperature of hot solution mixture was gradually lowered to 27 °C and allowed to settle at this temperature for a time period ranging from 60 minutes to precipitate of mucilaginous interfering impurities. The clarified supernatant phase was subjected to crystallization at 7 °C over a period of 12 hours. The crystalline oryzanol fraction was dried by vaccum drying to produce flakes, which was converted to fine free flowing powder by size reduction. The purity and yield of oryzanol obtained after crystallization are given in the following table. (Table Removed) Example! 100 gm of oryzanol enriched fraction (50 to 55 % oryzanol) unsaponifiable fraction [prepared as per Indira et al., (2002), patent #2] was mixed with suitable mixtures of organic solvents such as acetone and methanol (1: 3) at particular solid to solvent ratio of 1:5 and refluxed at elevated temperature of 80 °C for 1.25 hour to facilitate the complete dissolution of oryzanol throughout the solvent mixture. Then, the temperature of hot solution mixture was ally lowered to 25 °C and allowed to settle at this temperature for a time period ranging from 90 minutes to precipitate of mucilaginous interfering impurities. The clarified supernatant phase was subjected to crystallization at 7 °C over a period of 16 hours. The crystalline oryzanol fraction was dried by vacuum drying to produce flakes, which was converted to fine free flowing powder by size reduction. The purity and yield of oryzanol obtained after crystallization are given in the following table. (Table Removed) Advantages of the present invention 1. Process enables to obtain free flowing highly purified powder of oryzanol in much simpler manner. 2. Process is easy to scale-up and versatile enough to accommodate the changes in processing conditions. 3. Process enables partial fractionation of mucilaginous interfering impurities, which makes size reduction of final product easy. 4. Process enables to obtain an unsaponifiable fraction of rice bran oil soapstock devoid of acid and basic residues We claim: 1. A process for the preparation of oryzanol flakes from the oryzanol enriched saponifiable fraction of rice bran oil (RBO) soapstock wherein the oryzanol shows purity and yield as ranging between 60-65% and 70-75% respectively, the said process comprising steps of: (a) leaching the saponified and dehydrated rice bran oil soapstock to obtain oryzanol enriched unsaponified fraction, (b) dissolving the unsaponified fraction of step (a) in a mixture of organic solvent selected from a mixture of acetone and methanol in a ratio ranging between 1:3 to 1:4 at a reflux temperature. (c) cooling the mixture of step (b) gradually to a temperature in the range of 25 to 30°C. allowed to stand at this temperature for a period of 30 to 120 minutes to precipitate mucilaginous impurities, (d) separating the mucilaginous impurities of step (c) to obtain a clarified supernatant, (e) crystallizing the supernatant of step (d) at a temperature range of 5 to 10°C over a period of to to 16 hours to obtaincrystallized oryzanol, (f) separating and desolvating the crystalline oryzanol of step (e) to obtain flakes of oryzanol. 2. A process as claimed in claim 1. wherein the ratio of oryzanol enriched unsaponiliable fruction and organic solvent mixture is in the range of 1: 3 to 1: 6. 3. A process as claimed in claim 1. wherein in step (e). the crystallization may be perform adapting methods selected from temperature controlled fractional crystallization, temperation controlled antisolvent crystallization, antisolvent crystallization, high pressure gas antisolvent crystallization or combinations thereof. I A process as claimed in claim 1, wherein in step (f). desolvation is performed either by under vacuum or by distillation. 5.A process for the preparation of oryzanol flakes from the oryzanol enriched sapomfiable fraction of rice bran oil (RBO) soapstock substantially as herein describe with reference to examples accompanying this specification.

Documents:

408-DEL-2003-Abstract-(30-01-2009).pdf

408-del-2003-abstract.pdf

408-DEL-2003-Claims-(16-01-2009).pdf

408-DEL-2003-Claims-(30-01-2009).pdf

408-del-2003-claims.pdf

408-del-2003-complete specification (granted).pdf

408-DEL-2003-Correspondence-Others-(16-01-2009).pdf

408-DEL-2003-Correspondence-Others-(30-01-2009).pdf

408-del-2003-correspondence-others.pdf

408-del-2003-correspondence-po.pdf

408-DEL-2003-Description (Complete)-(16-01-2009).pdf

408-del-2003-description (complete)-30-01-2009.pdf

408-del-2003-description (complete).pdf

408-DEL-2003-Form-1-(16-01-2009).pdf

408-DEL-2003-Form-1-(30-01-2009).pdf

408-del-2003-form-1.pdf

408-del-2003-form-18.pdf

408-DEL-2003-Form-2-(30-01-2009).pdf

408-del-2003-form-2.pdf

408-DEL-2003-Form-3-(16-01-2009).pdf

408-del-2003-form-3.pdf

408-DEL-2003-Petition-137-(16-01-2009).pdf