Title of Invention	A PROCESS FOR PREPARATION OF ORYZANOL ENRICHED RICE BRAN OIL
Abstract	The present invention entitled "A Simple Two Step Alkali Treatment Process for the Production of Oryzanol Enriched Rice Bran Oil", is aimed at concentrating oryzanol by a solvent less process and recovery of spent oil for edible purposes. In this process, oryzanol enriched rice bran oil has been prepared starting from physically refined rice bran oil or crude rice bran oil through controlled alkali treatment and having greater than five fold enrichment in oryzanol for use in edible applications. The spent oil is recovered and can be reused, thereby all the starting oil can be utilized for food purposes. The other advantages of the process for industrial adoption are that there is no solvent usage for extraction of oryzanol into oil phase, and the main product and by-products can be re-used for edible/industrial purposes and the process can be adopted in the industry with minimal additional machinery.

Title of Invention

A PROCESS FOR PREPARATION OF ORYZANOL ENRICHED RICE BRAN OIL

Abstract

The present invention entitled "A Simple Two Step Alkali Treatment Process for the Production of Oryzanol Enriched Rice Bran Oil", is aimed at concentrating oryzanol by a solvent less process and recovery of spent oil for edible purposes. In this process, oryzanol enriched rice bran oil has been prepared starting from physically refined rice bran oil or crude rice bran oil through controlled alkali treatment and having greater than five fold enrichment in oryzanol for use in edible applications. The spent oil is recovered and can be reused, thereby all the starting oil can be utilized for food purposes. The other advantages of the process for industrial adoption are that there is no solvent usage for extraction of oryzanol into oil phase, and the main product and by-products can be re-used for edible/industrial purposes and the process can be adopted in the industry with minimal additional machinery.

Full Text	The present invention relates to "a process for the preparation of Oryzanol Enriched Rice Bran Oil". The invention is aimed at concentrating oryzanol by a solvent less process and recovery of spent oil for edible purposes. Therefore, in this invention a process for the preparation of oryzanol enriched rice bran oil starting from physically refined rice bran oil or crude rice bran oil subjected to controlled alkali treatment and having greater than five fold enrichment in oryzanol for use in edible applications. The spent oil is recovered and can be reused, thereby all the starting oil can be utilized for food purposes. The other advantages of the process for industrial adoption are that there is no solvent usage for extraction of oryzanol into oil phase, and the main product and by-products can be re-used for edible/industrial purposes and the process can be adopted in the industry with minimal additional machinery. Rice (oryza sativa L.) is an important cereal in many Asian Countries. India produces about 92 million tons of paddy annually [Vikas Singhal (Editor), Indian Agriculture, Indian Economic Data Research Centre, B-713, Panchvati, Vikaspuri, New Delhi, Chap.-8, 120-136, 2003]. The rice grain contains 5% bran and 12-18.5% oil in the bran and therefore it is a potential source of the oil. The potential availability of oil from rice bran is about 1 million tons in India and only 50% of it is being produced at present. The rice bran oil contains oryzanol (1-2%) which is an important micronutrient and is a mixture of ferulic acid esters unique to rice bran oil. It has many beneficial biological effects such as reduction of cholesterol in the blood, improvement of capillary action of blood vessel, anti-aging, anti-dandruff and anti-itching properties and patented products on these properties are produced in Japan, USA, and some other countries(Kamimura, M., S. Takahashi, and S. Sato. I"fluence of y-oryzanol on the skin capillary circulation, Bitamin 30:341(1964) [cited from Chem. Abstr. 62:57 83C (1965)]. Noboru, K., T. Yusho, Oryzanol containing cosmetics, Japanese Patent 70:32078 Oct. 16, 1970 [cited from Chem. Abstr, 74: 146249n (1977)], Shugo, M., Anti-dandruff and anti-itching Shampoo, Japanese Patent 79; 36306 March 17, 1979 [cited from Chem. Abstr, 91: 98764a, (1979)], Teruto, T., Dentifrices, Japanese Patent 78: 99339, Aug 30 (1978) [cited from Chem. Abstr, 89: 2042 48f (1978)], Hofman Cornelis, Van Amerongen Marnix P, and Zwanenburg Arend, Process for obtaining oryzanol, US patent, US2001047101, 29,November, 2001, Hisashi, I., et al., Production of oryzanol-rich rice bran oil, Japanese Patent, JP6340889, 13, December 1994]. .In the Indian context, we donot have any of these products, either in the form of concentrates or the pure oryzanol, but a number of patented processes and published literature are available. Rao et al(Rao, K.V.S.A., Rao, B.V.S.K., and Kaimal, T.N.B., Method for isolating oryzanol from soap stock of rice bran oil, International patent JP2002293793, 9,Oct.2002, and Rao, B.V.S. Rao, K.V.S.A., and Kaimal, T.N.B., Process for the isolation of oryzanols from rice bran oil soap-stock, Uspatent, US6410762, 25,June,2002) have patented the process of preparation of oryzanol from soap stock of rice bran oil both in India and the USA. In this process, the soap stock is treated with alkali and made into noodles which is extracted and purified to get oryzanol. The draw back with the process is that oryzanol also reacts with alkali to form soap and hence the decreased recovery as well as destruction of oryzanol may result. Das et al(Das, P.K., et al, Process for the isolation of oryzanols from crude dark acid oil(rice bran), international patent No.US5869708, dated 9, February 1999) have developed a process for the isolation of oryzanols from crude dark acid oil of rice bran through distillation of fatty acids by conventional methods followed by hydrolyzing the resultant residue and forming calcium ion aggregates of oryzanol followed by extraction and purification. The high temperatures normally used for the fatty acids distillation may degrade oryzanol and hence may not produce a good product. Gopala Krishna and Sakina Khatoon (Gopala Krishna, A.G., and Sakina Khatoon, A process for preparation of oryzanol concentrate from the rice bran oil soap-stock, Indian patent, 2150/DEL/98), and Gopala Krishna et al (Gopala Krishna, A.G., Sakina Khatoon and Indira, T.N., An improved process for preparation of oryzanol, Indian patent 398/DEL/03) have developed processes for the extraction and purification of oryzanol from soap stock by the partition technique. The extractability of oryzanol varied with presence of alkali in the solvent phases, making extraction of oryzanol difficult. Purnima Kaul et al (Purnima Kaul, T., Barhate, R.S., Indira, T.N., Narayan A.V., Prakash, V., Raghava Rao, K.S.M.S., and Appu Rao, A.G., Process for crystallization of oryzanol from oryzanol enriched fraction derived from rice bran oil soap stock. World Patent, WO2004055040, 1,July,2004) have developed a process for the crystallization of oryzanol from oryzanol enriched fraction of soap stock and the details are not available from the patent abstract. Singh (RP Singh, A process for the preparation/isolation of oryzanol compound from rice bran oil, Patent application No. 85/DEL/2003 dated 3.2.03 and source: The Gazette of India, April 19, 2003) has filed a patent on the isolation of oryzanol from rice bran oil (details of claims not available) and Seetharamaiah and Prabhakar [Seetharamaiah, G.S., & J.V.Prabhakar, Oryzanol content of rice bran oil and its extraction from soap-stock, J. Food Science & Technology,(lndia), vol.23, pp270-273(1986)] and Singh et al (R.P.Singh, R.K.Khanna, A.Mathur and R.Srivastava, Isolation of oryzanol concentrate from rice bran oil, J. Oil Technologists Association of India, vol.32, pp55-58(2000)have also published research papers on the subject. Both the researchers use a column chromatographic method for the isolation of oryzanol from crude rice bran oil followed bv diethyl ether elution which may be difficult to practice on a large scale as diethyl ether used in the method is low boiling, evaporation loss may be high, explosive, toxic and environment unfriendly. Hisashi et al (Hisashi, I., et al., Production of oryzanol rich rice bran oil, international patent, JP6340889, dated 13, December 1994) have developed a oryzanol rich rice bran oil from crude rice bran oil by removing fatty acids contained in it by steam distillation in a vacuum without using an alkali. The oil so obtained is similar to physically refined rice bran oils and the oryzanol content may not exceed 2-2.5%. Kenjiro (Fujita Kenjiro (JP) Method for producing oil from rice embroyo buds, international patent No. KR8601714, dated 18, October 1986) has patented a procedure for the production of a high pressure pressed rice germ oil without treating with chemicals. The oil may not have high oryzanol levels and may not exceed 2-2.5% as in an earlier patent which may be slightly better than the physical refined rice bran oil in its oryzanol content. Object of the invention: The object of this invention is to provide a process for the preparation of oryzanol enriched rice bran oil starting from physically refined rice bran oil or crude rice bran oil subjected to controlled alkali treatment and having greater than five fold enrichment in oryzanol for use in edible applications. So far, there is no published literature or patented process for the production of greater than five fold oryzanol enriched rice bran oil. Another object of the invention is to convert the spent rice bran oil for edible purposes. The salient features of the process include the following steps: ^Treatment with (first stage) alkali for neutralization of real ft.; ii)Separation of soap layer and collection of oil layer iii)Treatment of oil layer with (second stage) stronger alkali for extraction of oryzanol into alkali phase iv)Separation of oryzanol containing bottom layer and top oil layer. v)Acidification (both the layers separately). vi)Washing (both the layers separately). vii)Drying under vacuum (both the layers separately) viii)Obtain oryzanol enriched rice bran oil and refined rice bran oil and byproducts are soap and salt xi)Process loss : 2-10% x)The advantages of the process are that there is no solvent usage for extraction of oryzanol into oil phase, and the main product and byproducts can be re-used for edible/industrial purposes. The process can be adopted in the industry with minimal additional machinery. Accordingly the present invention relates to a process for the preparation of Oryzanol Enriched Rice Bran Oil" which comprises the steps of: a. subjecting rice bran oil to alkali treatment with a standard alkali solution equivalent to the real free fatty acids content, selected from sodium hydroxide, sodium carbonate, potassium hydroxide, and calcium hydroxide b. mixing at 250 - 450 RPM for a period of 10 - 30 minutes, c. settling for a period of 1 hr to 48hrs for separation of bottom soap layer and top oil layer, d. separating top oil layer containing oryzanol and bottom layer containing soap and rejecting the bottom soap layer a:? soap stock, e. treating the top oil layer at the controlled alkali treatment with a lower strength stronger alkali solution selected from sodium hydroxide and potassium hydroxide at a rate of 5-6% f. mixing at 250 - 450 RPM for a period of 10 - 30 minutes, g. settling for a period of 1 hr to 48hrs for separation of bottom layer and top oil layer, h. separating top layer and the bottom layer, i. treating of bottom layers with dilute acid, selected from hydrochloric, sulphuric, acetic or citric acids, j. washing the excess acid with water followed by drying under vacuum to obtain the oryzanol enriched rice bran oil. k. treating top layer (oil) with dilute acid and washing with hot water followed by drying under vacuum to recover the spent oil for edible purposes. In an embodiment of the present invention, the alkali used has a strength of minimum of 10%, In an embodiment of the present invention, the mixing temperature is ranging from 15degree centigrade to 60 degree centigrade, In an embodiment of the present invention, the starting oil is crude, degummed or dewaxed or physically refined rice bran oils, In an embodiment of the present invention, the oryzanol enriched rice bran oil has a oryzanol content of 3-15%, having the 24-methylene cycloartanyl ferulate content of 10-60%, Process Description: The Process of Preparation of Oryzanol Enriched Rice Bran Oil is described herewith as follows: Physically Refined Rice Bran Oil (7.5 Kg) of 0.77% ffa content (real ffa content = 0.39%) and 0.88% oryzanol content was taken in a suitable container, treated at the first step alkali treatment stage with sodium hydroxide solution (35 ml of 20 degree Baume) equivalent to the real free fatty acids content(calculated by using the formula real ffa = total ffa minus (%Oryzanol X 0.425) (0.425 is the factor for the ffa content contributed by the acidity of oryzanol) and stirred at 250RPM at room temperature for 20 minutes and allowed to settle for 16hrs to settle the soap as bottom aqueous layer. The top oil layer is separated and preserved for further processing, and the bottom soap layer is rejected as soapstock. In the next step, the top oil layer(7.2kg) is taken for the second step controlled alkali treatment stage and treated with 450ml potassium hydroxide (10% solution) and stirred at 450RPM at room temperature for 20minutes. The contents were allowed to settle for 2 hours at the same temperature, afterwards, decanted top layer (oil layer) and the bottom layer (oryzanol enriched layer) was collected separately. The bottom layer was acidified with 333ml (2N hydrochloric acid) to get an oryzanol enriched oily layer and is washed two times with water; and all the aqueous washings collected. The oily layer was dried under vacuum and the washings were added to the first decanted recovered oil (top layer) and stirred well and the aqueous layer rejected and the recovered oil layer was washed with hot water (80°C), three times. The aqueous layers (rejected layers) were pooled and sun dried to get potassium chloride(salt). The washed recovered oil was dried under vacuum to get spent rice bran oil for use as refined oil for edible purposes. Similarly, Crude Rice Bran Oil(1000g) is degummed and dewaxed by known methods to get dewaxed rice bran oil(954g) and the dewaxed oil was treated at the first step alkali treatment stage with sodium hydroxide solution (68.7ml of 20° Baume) equivalent to the real free fatty acids content(calculated by using the formula real ffa = total ffa minus (%Oryzanol X 0.425) (0.425 is the factor for the ffa content contributed by the acidity of oryzanol) and stirred at 250RPM at room temperature for 20 minutes and allowed to settle for 16hrs to settle the soap as bottom aqueous layer. The top oil layer is separated and preserved for further processing, and the bottom soap layer is rejected as soapstock. In the next step, the top oil layer(595g) is taken for the second step controlled alkali treatment stage and treated with 35.7ml potassium hydroxide(10%) and stirred at 250RPM at room temperature for 20minutes. The contents were allowed to settle for 2 hours at the same temperature, afterwards, decanted top layer (oil layer) and the bottom layer (oryzanol enriched layer) was collected separately. The bottom layer was acidified with 26.4ml (2N hydrochloric acid) to get an oryzanol enriched oily layer and is washed two times with water; and all the aqueous washings collected. The oryzanol enriched oily layer was dried under vacuum and the washings were added to the first decanted recovered oil (top layer) and stirred well and the aqueous layer rejected and the recovered oil layer was washed with hot water (80°C), three times. The aqueous layers (rejected layers) were pooled and sun dried to get potassium chloride(salt). The washed recovered oil was dried under vacuum to get spent rice bran oil for use as refined oil for edible purposes. Physically refined Rice Bran Oil (7.5kg) + NaOH solution(35ml) (Figure Removed) The novelty in this invention is that the real free fatty acids are carefully neutralized with alkali in the first stage and later in the second stage, the ffa arising out of the acidity contributed by oryzanol is neutralized using a lesser strength stronger alkali to give an aqueous layer containing oryzanol in the form of alkali metal derivative which after neutralization with an acidic material and washing, goes into the oil phase and forms oryzanol enriched oil layer which is the invention in the process. The spent oil is recovered and can be reused, thereby all the starting oil can be utilized for food purposes and also a new product has been developed in the invention. The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention(Table 1-4). In the experiments cited under examples 1-5, the first stage alkali neutralization was not included as the quantity of oil handled were very small (10-500g ) and were physically refined rice bran oils with very low ffa content. Therefore, only batch sizes of 1000g and above include two step alkali treatments (examples 6 & 7). Example-1 1. Started with 10 g physical refined rice bran oil (PRBO), 0.5 ml KOH (10%) was added and stirred at 28°C (r.t) for 20 min 2. Allowed to settle for 14 hrs 3. Two layers formed and were separated by decantation, top layer (oil), bottom layer ( oryzanol enriched aqueous layer) 4. Bottom layer was acidified with 2N HCI and washed once with water. 5. Dried under vacuum at 80°C for 10-12 hrs to get a natural oryzanol enriched rice bran oil ready for use in foods having a desirable oryzanol content greater than five times the starting rice bran oil. 6. The washings from processing of bottom layer (above) was added to the top layer(oily layer) and mixed, and aqueous layer rejected, the recovered oil layer washed with hot water (80°C) three times and the oil layer separated. 7. The oil layer was dried under vacuum at 80°C for 10 hrs to get original cooking oil having oryzanol content of 0.3-0.4%. 8. Aqueous layer sun dried for a week to get potassium chloride (salt) Example -2 1. Started with 25 g physical refined rice bran oil (PRBO), 1.25 ml KOH (10%) was added and stirred for 20 min at 28° C (r.t.) 2. Allow to settle for 15 hrs 3. Two layers were separated by decantation, top layer (oil) and bottom layer (oryzanol enriched aqueous layer) 4. Bottom layer was acidified with 2 N HCI and washed once with water. 5. Dried under vacuum at 80°C for 10-12 hrs to get a natural oryzanol enriched rice bran oil ready for use in foods having a desirable oryzanol content greater than five times the starting rice bran oil 6. Bottom layer washed aqueous layer was added to top layer and washed with hot water (80°C) three times and the oil layer separated. 7. Oil layer was dried under vacuum at 80°C for 1-2 hrs to get original cooking oil having oryzanol content of 0.3-0.4% 8. Aqueous layer sun dried for a week to get potassium chloride (salt) Example - 3 1. Started with 100 g physical refined rice bran oil (PRBO), 5.0ml KOH (10%) was added and stirred for 20 min at 28° C (r.t.) 2. Allow to settle for 15 hrs 3. Two layers were separated by decantation, top layer (oil) and bottom layer (oryzanol enriched aqueous layer) 4. Bottom layer was acidified with 2 N HCI and washed once with water 5. Dried under vacuum at 80°C for 10-12 hrs to get a natural oryzanol enriched rice bran oil ready for use in foods having a desirable oryzanol content greater than five times the starting rice bran oil 6. Bottom layer washed aqueous layer was added to top layer and washed with hot water (80°C) three times - oil layer 7. Oil layer was dried under vacuum at 80°C for 1-2 hrs to get original cooking oil having oryzanol content of 0.3-0.4% 8. Aqueous layer sun dried for a week to get potassium chloride (salt) Example - 4 1. Started with 500 g physical refined rice bran oil (PRBO), 25ml KOH (10%) was added and stirred for 20 min at 29° C (r.t.) 2. Allow to settle for 16 hrs 3. Two layers were separated by decantation, top layer (oil) and bottom layer (oryzanol enriched aqueous layer) 4. Bottom layer was acidified with 2 N HCI and washed once with water 5. Dried under vacuum at 80°C for 10-12 hrs to get a natural oryzanol enriched rice bran oil ready for use in foods having a desirable oryzanol content greater than five times the starting rice bran oil 6. Bottom layer washed aqueous layer was added to top layer and washed with hot water (80°C) three times and the oil layer separated. 7. The oil layer was dried under vacuum at 80°C for 1-2 hrs to get original cooking oil having oryzanol content of 0.3-0.4% 8. Aqueous layer was sun dried for a week to get potassium chloride (salt) Example - 5 1. Started with 1 kg physical refined rice bran oil (PRBO), 60ml KOH (10% solution) and stirred for 20 min at 30°C (r.t.) & 450 RPM 2. Allow to settle for 2 hrs 3. Two layers were separated by decantation, top layer oil and bottom layer (oryzanol enriched aqueous layer) 4. Bottom layer was acidified with 2 N HCI (44.4 ml) 5. Washed with water two times. 6. Dried under vacuum at 80°C for 10-12 hrs to get a natural oryzanol enriched rice bran oil ready for use in foods having a desirable oryzanol content greater than five times the starting rice bran oil 7. Top layer was washed with hot water (80°C) until it is free from soap 8. Oil layer was dried under vacuum at 80°C for 1-2 hrs to get original cooking oil having oryzanol content of 0.3-0.4%. 9. Aqueous layer sun dried for a week to get potassium chloride (salt) Example-6 1. For the first stage alkali neutralization, started with 7.5kg physically refined rice bran oil(PRBO), treated with 35ml of NaOH, 20° Baume solution, stirred at 30° C and at 450RPM for 20 min 2. Allow to settle for 16 hours and decant the supernatant oil and reject bottom soap layer. 3. For the second stage oryzanol extraction, started with 7.2 kg of rice bran oil, 450ml KOH (10%) and stirred at 30°C (r.t.) and at 450RPM for 20 min 4. Allow to settle for 2 hrs 5. Two layers were separated by decantation, top layer (oil) and bottom layer (oryzanol enriched aqueous layer) 6. Bottom layer was acidified with 2 N HCI (333 ml) 7. Washed with water two times. 8. Dried under vacuum at 80°C for 10-12 hrs to get a natural oryzanol enriched rice bran oil ready for use in foods having a desirable oryzanol content greater than five times the starting rice bran oil 9. Top layer was washed with hot water (80°C) until it is free from soap 10. Oil layer was dried under vacuum at 80°C for 1-2 hrs to get original cooking oil having oryzanol content of 0.3-0.4%. 11 .Aqueous layer was sun dried for a week to get potassium chloride (salt) Example - 7 1. Started with 954 g of degummed and dewaxed rice bran oil (RBO), 68.7ml NaOH (20°Be) and stirred at 250RPM at 29°C (r.t.) for 20 min, 2. Allowed to settle for 16 hrs 3. Two layers were separated by decantation, top layer (oil) and bottom layer (soap layer) 4. The top oil layer(595g) was treated with 35.7ml KOH(10%) and stirred at 250RPM at 29°C for 20 min, 5. Allowed to settle for 16 hrs, 6. Two layers were separated by decantation, top layer (oil) and bottom layer (oryzanol enriched aqueous layer) 7. Bottom layer was acidified with 2 N HCI(26.4ml) and washed once with water 8. Dried under vacuum at 80°C for 10-12 hrs to get a natural oryzanol enriched rice bran oil ready for use in foods having a desirable oryzanol content greater than five times the starting rice bran oil 9. Top layer was washed with hot water 10. The oil layer was dried under vacuum at 80°C for 1-2 hrs to get original cooking oil having oryzanol content of 0.3-0.4% 11 .Aqueous layer was sun dried for a week to get potassium chloride (salt) Table-1: Preparation of oryzanol enriched rice bran oil (OZ-RBO) (Table Removed) Table-2: Characteristics of the oryzanol enriched rice bran oil (Table Removed) Table-3: Recovery of rice bran oil after processing (Table Removed) Table-4: Characteristics of oil (after removing Oryzanol enriched fraction) (Table Removed) Advantages: The advantages of the invention are that a rice bran oil enriched with oryzanol to the extent of greater than five fold the content present in the starting rice bran oil has been prepared in the invention without using any organic solvent and the oryzanol enriched rice bran oil can be straight away u»ed for application in foods at desired levels. So far only oryzanol concentrates were available having 30-40% oryzanol along with the remaining 60-70% fatty material which may contain toxic solvent residues, and oxidized fatty matter arising out of long storage of soapstocks or drastic treatments during molecular distillation and other procedures adopted for the preparation of oryzanol concentrates, but the oryzanol enriched rice bran oil prepared in this invention contains good quality rice bran oil free of oxidized fatty material. The other advantages of the process for industrial adoption are that there is no solvent usage for extraction of oryzanol into oil phase, and the main product and byproducts can be re-used for edible/industrial purposes and the process can be adopted in the industry with minimal additional machinery. We claim: 1. A process for the preparation of Oryzanol Enriched Rice Bran Oil" which comprises the steps of: a. subjecting rice bran oil to alkali treatment with a standard alkali solution equivalent to the real free fatty acids content, selected from sodium hydroxide, sodium carbonate, potassium hydroxide, and calcium hydroxide b. mixing at 250 - 450 RPM for a period of 10 - 30 minutes, c. settling for a period of 1 hr to 48hrs for separation of bottom soap layer and top oil layer, d. separating top oil layer containing oryzanol and bottom layer containing soap and rejecting the bottom soap layer as soap stock, e. treating the top oil layer at the controlled alkali treatment with a lower strength stronger alkali solution selected from sodium hydroxide and potassium hydroxide at a rate of 5-6% f. mixing at 250 - 450 RPM for a period of 10 - 30 minutes, g. settling for a period of 1 hr to 48hrs for separation of bottom layer and top oil layer, h. separating top layer and the bottom layer, i. treating of bottom layers with dilute acid, selected from hydrochloric, sulphuric, acetic or citric acids, j. washing the excess acid with water followed by drying under vacuum to obtain the oryzanol enriched rice bran oil. k. treating top layer (oil) with dilute acid and washing with hot water followed by drying under vacuum to recover the spent oil for edible purposes. 2. A process as claimed in claim 1, above, the alkali used has a strength of minimum of 10%, 3. A process as claimed in claims 1 & 2, the mixing temperature is ranging from 15degree centigrade to 60 degree centigrade, 4. As claimed in claims 1 to 3, above, the starting oil is crude, degummed or dewaxed or physically refined rice bran oils, 5. As claimed in claims 1 to 4, above, the oryzanol enriched rice bran oil has a oryzanol content of 3-15%, having the 24-methylene cycloartanyl ferulate content of 10-60%, 6. A process for the preparation of Oryzanol Enriched Rice Bran Oil substantially as herein described with reference to examples accompanying this specification.

Full Text

The present invention relates to "a process for the preparation of Oryzanol Enriched Rice Bran Oil".
The invention is aimed at concentrating oryzanol by a solvent less process and recovery of spent oil for edible purposes. Therefore, in this invention a process for the preparation of oryzanol enriched rice bran oil starting from physically refined rice bran oil or crude rice bran oil subjected to controlled alkali treatment and having greater than five fold enrichment in oryzanol for use in edible applications. The spent oil is recovered and can be reused, thereby all the starting oil can be utilized for food purposes. The other advantages of the process for industrial adoption are that there is no solvent usage for extraction of oryzanol into oil phase, and the main product and by-products can be re-used for edible/industrial purposes and the process can be adopted in the industry with minimal additional machinery.
Rice (oryza sativa L.) is an important cereal in many Asian Countries. India produces about 92 million tons of paddy annually [Vikas Singhal (Editor), Indian Agriculture, Indian Economic Data Research Centre, B-713, Panchvati, Vikaspuri, New Delhi, Chap.-8, 120-136, 2003]. The rice grain contains 5% bran and 12-18.5% oil in the bran and therefore it is a potential source of the oil. The potential availability of oil from rice bran is about 1 million tons in India and only 50% of it is being produced at present. The rice bran oil contains oryzanol (1-2%) which is an important micronutrient and is a mixture of ferulic acid esters unique to rice bran oil. It has many beneficial biological effects such as reduction of cholesterol in the blood, improvement of capillary action of blood vessel, anti-aging, anti-dandruff and anti-itching properties and patented products on these properties are produced in Japan, USA, and some other countries(Kamimura, M., S. Takahashi, and S. Sato. I"fluence of y-oryzanol on the skin capillary circulation, Bitamin 30:341(1964) [cited from

Chem. Abstr. 62:57 83C (1965)]. Noboru, K., T. Yusho, Oryzanol containing cosmetics, Japanese Patent 70:32078 Oct. 16, 1970 [cited from Chem. Abstr, 74: 146249n (1977)], Shugo, M., Anti-dandruff and anti-itching Shampoo, Japanese Patent 79; 36306 March 17, 1979 [cited from Chem. Abstr, 91: 98764a, (1979)], Teruto, T., Dentifrices, Japanese Patent 78: 99339, Aug 30 (1978) [cited from Chem. Abstr, 89: 2042 48f (1978)], Hofman Cornelis, Van Amerongen Marnix P, and Zwanenburg Arend, Process for obtaining oryzanol, US patent, US2001047101, 29,November, 2001, Hisashi, I., et al., Production of oryzanol-rich rice bran oil, Japanese Patent, JP6340889, 13, December 1994].
.In the Indian context, we donot have any of these products, either in the form of concentrates or the pure oryzanol, but a number of patented processes and published literature are available. Rao et al(Rao, K.V.S.A., Rao, B.V.S.K., and Kaimal, T.N.B., Method for isolating oryzanol from soap stock of rice bran oil, International patent JP2002293793, 9,Oct.2002, and Rao, B.V.S. Rao, K.V.S.A., and Kaimal, T.N.B., Process for the isolation of oryzanols from rice bran oil soap-stock, Uspatent, US6410762, 25,June,2002) have patented the process of preparation of oryzanol from soap stock of rice bran oil both in India and the USA. In this process, the soap stock is treated with alkali and made into noodles which is extracted and purified to get oryzanol. The draw back with the process is that oryzanol also reacts with alkali to form soap and hence the decreased recovery as well as destruction of oryzanol may result. Das et al(Das, P.K., et al, Process for the isolation of oryzanols from crude dark acid oil(rice bran), international patent No.US5869708, dated 9, February 1999) have developed a process for the isolation of oryzanols from crude dark acid oil of rice bran through distillation of fatty acids by conventional methods followed by hydrolyzing the resultant residue and forming calcium ion aggregates of oryzanol followed by extraction and purification. The high

temperatures normally used for the fatty acids distillation may degrade oryzanol and hence may not produce a good product.
Gopala Krishna and Sakina Khatoon (Gopala Krishna, A.G., and Sakina Khatoon, A process for preparation of oryzanol concentrate from the rice bran oil soap-stock, Indian patent, 2150/DEL/98), and Gopala Krishna et al (Gopala Krishna, A.G., Sakina Khatoon and Indira, T.N., An improved process for preparation of oryzanol, Indian patent 398/DEL/03) have developed processes for the extraction and purification of oryzanol from soap stock by the partition technique. The extractability of oryzanol varied with presence of alkali in the solvent phases, making extraction of oryzanol difficult. Purnima Kaul et al (Purnima Kaul, T., Barhate, R.S., Indira, T.N., Narayan A.V., Prakash, V., Raghava Rao, K.S.M.S., and Appu Rao, A.G., Process for crystallization of oryzanol from oryzanol enriched fraction derived from rice bran oil soap stock. World Patent, WO2004055040, 1,July,2004) have developed a process for the crystallization of oryzanol from oryzanol enriched fraction of soap stock and the details are not available from the patent abstract. Singh (RP Singh, A process for the preparation/isolation of oryzanol compound from rice bran oil, Patent application No. 85/DEL/2003 dated 3.2.03 and source: The Gazette of India, April 19, 2003) has filed a patent on the isolation of oryzanol from rice bran oil (details of claims not available) and Seetharamaiah and Prabhakar [Seetharamaiah, G.S., & J.V.Prabhakar, Oryzanol content of rice bran oil and its extraction from soap-stock, J. Food Science & Technology,(lndia), vol.23, pp270-273(1986)] and Singh et al (R.P.Singh, R.K.Khanna, A.Mathur and R.Srivastava, Isolation of oryzanol concentrate from rice bran oil, J. Oil Technologists Association of India, vol.32, pp55-58(2000)have also published research papers on the subject. Both the researchers use a column chromatographic method for the isolation of oryzanol from crude rice bran oil followed bv diethyl ether elution which may be difficult to practice on a large scale as diethyl ether

used in the method is low boiling, evaporation loss may be high, explosive, toxic and environment unfriendly.
Hisashi et al (Hisashi, I., et al., Production of oryzanol rich rice bran oil, international patent, JP6340889, dated 13, December 1994) have developed a oryzanol rich rice bran oil from crude rice bran oil by removing fatty acids contained in it by steam distillation in a vacuum without using an alkali. The oil so obtained is similar to physically refined rice bran oils and the oryzanol content may not exceed 2-2.5%. Kenjiro (Fujita Kenjiro (JP) Method for producing oil from rice embroyo buds, international patent No. KR8601714, dated 18, October 1986) has patented a procedure for the production of a high pressure pressed rice germ oil without treating with chemicals. The oil may not have high oryzanol levels and may not exceed 2-2.5% as in an earlier patent which may be slightly better than the physical refined rice bran oil in its oryzanol content.
Object of the invention:
The object of this invention is to provide a process for the preparation of oryzanol enriched rice bran oil starting from physically refined rice bran oil or crude rice bran oil subjected to controlled alkali treatment and having greater than five fold enrichment in oryzanol for use in edible applications. So far, there is no published literature or patented process for the production of greater than five fold oryzanol enriched rice bran oil. Another object of the invention is to convert the spent rice bran oil for edible purposes.
The salient features of the process include the following steps: ^Treatment with (first stage) alkali for neutralization of real ft.;

ii)Separation of soap layer and collection of oil layer
iii)Treatment of oil layer with (second stage) stronger alkali for extraction of oryzanol into alkali phase
iv)Separation of oryzanol containing bottom layer and top oil layer.
v)Acidification (both the layers separately).
vi)Washing (both the layers separately).
vii)Drying under vacuum (both the layers separately)
viii)Obtain oryzanol enriched rice bran oil and refined rice bran oil and byproducts are soap and salt
xi)Process loss : 2-10%
x)The advantages of the process are that there is no solvent usage for extraction of oryzanol into oil phase, and the main product and byproducts can be re-used for edible/industrial purposes. The process can be adopted in the industry with minimal additional machinery.
Accordingly the present invention relates to a process for the preparation of Oryzanol Enriched Rice Bran Oil" which comprises the steps of:
a. subjecting rice bran oil to alkali treatment with a standard alkali
solution equivalent to the real free fatty acids content, selected from
sodium hydroxide, sodium carbonate, potassium hydroxide, and
calcium hydroxide
b. mixing at 250 - 450 RPM for a period of 10 - 30 minutes,
c. settling for a period of 1 hr to 48hrs for separation of bottom soap
layer and top oil layer,
d. separating top oil layer containing oryzanol and bottom layer
containing soap and rejecting the bottom soap layer a:? soap stock,

e. treating the top oil layer at the controlled alkali treatment with a
lower strength stronger alkali solution selected from sodium
hydroxide and potassium hydroxide at a rate of 5-6%
f. mixing at 250 - 450 RPM for a period of 10 - 30 minutes,
g. settling for a period of 1 hr to 48hrs for separation of bottom layer
and top oil layer,
h. separating top layer and the bottom layer,
i. treating of bottom layers with dilute acid, selected from hydrochloric,
sulphuric, acetic or citric acids, j. washing the excess acid with water followed by drying under
vacuum to obtain the oryzanol enriched rice bran oil. k. treating top layer (oil) with dilute acid and washing with hot water
followed by drying under vacuum to recover the spent oil for edible
purposes.
In an embodiment of the present invention, the alkali used has a strength of minimum of 10%,
In an embodiment of the present invention, the mixing temperature is ranging from 15degree centigrade to 60 degree centigrade,
In an embodiment of the present invention, the starting oil is crude, degummed or dewaxed or physically refined rice bran oils,
In an embodiment of the present invention, the oryzanol enriched rice bran oil has a oryzanol content of 3-15%, having the 24-methylene cycloartanyl ferulate content of 10-60%,
Process Description:
The Process of Preparation of Oryzanol Enriched Rice Bran Oil is described herewith as follows:

Physically Refined Rice Bran Oil (7.5 Kg) of 0.77% ffa content (real ffa content = 0.39%) and 0.88% oryzanol content was taken in a suitable container, treated at the first step alkali treatment stage with sodium hydroxide solution (35 ml of 20 degree Baume) equivalent to the real free fatty acids content(calculated by using the formula real ffa = total ffa minus (%Oryzanol X 0.425) (0.425 is the factor for the ffa content contributed by the acidity of oryzanol) and stirred at 250RPM at room temperature for 20 minutes and allowed to settle for 16hrs to settle the soap as bottom aqueous layer. The top oil layer is separated and preserved for further processing, and the bottom soap layer is rejected as soapstock. In the next step, the top oil layer(7.2kg) is taken for the second step controlled alkali treatment stage and treated with 450ml potassium hydroxide (10% solution) and stirred at 450RPM at room temperature for 20minutes. The contents were allowed to settle for 2 hours at the same temperature, afterwards, decanted top layer (oil layer) and the bottom layer (oryzanol enriched layer) was collected separately. The bottom layer was acidified with 333ml (2N hydrochloric acid) to get an oryzanol enriched oily layer and is washed two times with water; and all the aqueous washings collected. The oily layer was dried under vacuum and the washings were added to the first decanted recovered oil (top layer) and stirred well and the aqueous layer rejected and the recovered oil layer was washed with hot water (80°C), three times. The aqueous layers (rejected layers) were pooled and sun dried to get potassium chloride(salt). The washed recovered oil was dried under vacuum to get spent rice bran oil for use as refined oil for edible purposes.
Similarly, Crude Rice Bran Oil(1000g) is degummed and dewaxed by known methods to get dewaxed rice bran oil(954g) and the dewaxed oil was treated at the first step alkali treatment stage with sodium hydroxide solution (68.7ml of 20° Baume) equivalent to the real free fatty acids content(calculated by using the formula real ffa = total ffa minus

(%Oryzanol X 0.425) (0.425 is the factor for the ffa content contributed by the acidity of oryzanol) and stirred at 250RPM at room temperature for 20 minutes and allowed to settle for 16hrs to settle the soap as bottom aqueous layer. The top oil layer is separated and preserved for further processing, and the bottom soap layer is rejected as soapstock. In the next step, the top oil layer(595g) is taken for the second step controlled alkali treatment stage and treated with 35.7ml potassium hydroxide(10%) and stirred at 250RPM at room temperature for 20minutes. The contents were allowed to settle for 2 hours at the same temperature, afterwards, decanted top layer (oil layer) and the bottom layer (oryzanol enriched layer) was collected separately. The bottom layer was acidified with 26.4ml (2N hydrochloric acid) to get an oryzanol enriched oily layer and is washed two times with water; and all the aqueous washings collected. The oryzanol enriched oily layer was dried under vacuum and the washings were added to the first decanted recovered oil (top layer) and stirred well and the aqueous layer rejected and the recovered oil layer was washed with hot water (80°C), three times. The aqueous layers (rejected layers) were pooled and sun dried to get potassium chloride(salt). The washed recovered oil was dried under vacuum to get spent rice bran oil for use as refined oil for edible purposes.

Physically refined Rice Bran Oil (7.5kg) + NaOH solution(35ml)
(Figure Removed)
The novelty in this invention is that the real free fatty acids are carefully neutralized with alkali in the first stage and later in the second stage, the ffa arising out of the acidity contributed by oryzanol is neutralized using a lesser strength stronger alkali to give an aqueous layer containing oryzanol in the form of alkali metal derivative which after neutralization with an acidic material and washing, goes into the oil phase and forms oryzanol enriched oil layer which is the invention in the process. The spent oil is recovered and can be reused, thereby all the starting oil can be utilized for food purposes and also a new product has been developed in the invention.
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention(Table 1-4). In the experiments cited under examples 1-5, the first stage alkali neutralization was not included as the quantity of oil handled were very small (10-500g ) and were physically refined rice bran oils with very low ffa content. Therefore, only batch sizes of 1000g and above include two step alkali treatments (examples 6 & 7).
Example-1
1. Started with 10 g physical refined rice bran oil (PRBO), 0.5 ml KOH (10%) was added and stirred at 28°C (r.t) for 20 min
2. Allowed to settle for 14 hrs
3. Two layers formed and were separated by decantation, top layer (oil), bottom layer ( oryzanol enriched aqueous layer)
4. Bottom layer was acidified with 2N HCI and washed once with water.

5. Dried under vacuum at 80°C for 10-12 hrs to get a natural oryzanol enriched rice bran oil ready for use in foods having a desirable oryzanol content greater than five times the starting rice bran oil.
6. The washings from processing of bottom layer (above) was added to the top layer(oily layer) and mixed, and aqueous layer rejected, the recovered oil layer washed with hot water (80°C) three times and the oil layer separated.
7. The oil layer was dried under vacuum at 80°C for 10 hrs to get original cooking oil having oryzanol content of 0.3-0.4%.
8. Aqueous layer sun dried for a week to get potassium chloride (salt)
Example -2
1. Started with 25 g physical refined rice bran oil (PRBO), 1.25 ml KOH (10%) was added and stirred for 20 min at 28° C (r.t.)
2. Allow to settle for 15 hrs
3. Two layers were separated by decantation, top layer (oil) and bottom layer (oryzanol enriched aqueous layer)
4. Bottom layer was acidified with 2 N HCI and washed once with water.
5. Dried under vacuum at 80°C for 10-12 hrs to get a natural oryzanol enriched rice bran oil ready for use in foods having a desirable oryzanol content greater than five times the starting rice bran oil
6. Bottom layer washed aqueous layer was added to top layer and washed with hot water (80°C) three times and the oil layer separated.

7. Oil layer was dried under vacuum at 80°C for 1-2 hrs to get original cooking oil having oryzanol content of 0.3-0.4%
8. Aqueous layer sun dried for a week to get potassium chloride (salt)
Example - 3
1. Started with 100 g physical refined rice bran oil (PRBO), 5.0ml KOH (10%) was added and stirred for 20 min at 28° C (r.t.)
2. Allow to settle for 15 hrs
3. Two layers were separated by decantation, top layer (oil) and bottom layer (oryzanol enriched aqueous layer)
4. Bottom layer was acidified with 2 N HCI and washed once with water
5. Dried under vacuum at 80°C for 10-12 hrs to get a natural oryzanol enriched rice bran oil ready for use in foods having a desirable oryzanol content greater than five times the starting rice bran oil
6. Bottom layer washed aqueous layer was added to top layer and washed with hot water (80°C) three times - oil layer
7. Oil layer was dried under vacuum at 80°C for 1-2 hrs to get original cooking oil having oryzanol content of 0.3-0.4%
8. Aqueous layer sun dried for a week to get potassium chloride (salt)
Example - 4
1. Started with 500 g physical refined rice bran oil (PRBO), 25ml KOH (10%) was added and stirred for 20 min at 29° C (r.t.)
2. Allow to settle for 16 hrs
3. Two layers were separated by decantation, top layer (oil) and bottom layer (oryzanol enriched aqueous layer)

4. Bottom layer was acidified with 2 N HCI and washed once with water
5. Dried under vacuum at 80°C for 10-12 hrs to get a natural oryzanol enriched rice bran oil ready for use in foods having a desirable oryzanol content greater than five times the starting rice bran oil
6. Bottom layer washed aqueous layer was added to top layer and washed with hot water (80°C) three times and the oil layer separated.
7. The oil layer was dried under vacuum at 80°C for 1-2 hrs to get original cooking oil having oryzanol content of 0.3-0.4%
8. Aqueous layer was sun dried for a week to get potassium chloride (salt)
Example - 5
1. Started with 1 kg physical refined rice bran oil (PRBO), 60ml KOH (10% solution) and stirred for 20 min at 30°C (r.t.) & 450 RPM
2. Allow to settle for 2 hrs
3. Two layers were separated by decantation, top layer oil and bottom layer (oryzanol enriched aqueous layer)

4. Bottom layer was acidified with 2 N HCI (44.4 ml)
5. Washed with water two times.
6. Dried under vacuum at 80°C for 10-12 hrs to get a natural oryzanol enriched rice bran oil ready for use in foods having a desirable oryzanol content greater than five times the starting rice bran oil
7. Top layer was washed with hot water (80°C) until it is free from soap

8. Oil layer was dried under vacuum at 80°C for 1-2 hrs to get original cooking oil having oryzanol content of 0.3-0.4%.
9. Aqueous layer sun dried for a week to get potassium chloride (salt)
Example-6
1. For the first stage alkali neutralization, started with 7.5kg physically refined rice bran oil(PRBO), treated with 35ml of NaOH, 20° Baume solution, stirred at 30° C and at 450RPM for 20 min
2. Allow to settle for 16 hours and decant the supernatant oil and reject bottom soap layer.
3. For the second stage oryzanol extraction, started with 7.2 kg of rice bran oil, 450ml KOH (10%) and stirred at 30°C (r.t.) and at 450RPM for 20 min
4. Allow to settle for 2 hrs
5. Two layers were separated by decantation, top layer (oil) and bottom layer (oryzanol enriched aqueous layer)
6. Bottom layer was acidified with 2 N HCI (333 ml)
7. Washed with water two times.
8. Dried under vacuum at 80°C for 10-12 hrs to get a natural oryzanol enriched rice bran oil ready for use in foods having a desirable oryzanol content greater than five times the starting rice bran oil
9. Top layer was washed with hot water (80°C) until it is free from soap
10. Oil layer was dried under vacuum at 80°C for 1-2 hrs to get original cooking oil having oryzanol content of 0.3-0.4%.
11 .Aqueous layer was sun dried for a week to get potassium chloride (salt)

Example - 7
1. Started with 954 g of degummed and dewaxed rice bran oil (RBO), 68.7ml NaOH (20°Be) and stirred at 250RPM at 29°C (r.t.) for 20 min,
2. Allowed to settle for 16 hrs
3. Two layers were separated by decantation, top layer (oil) and bottom layer (soap layer)
4. The top oil layer(595g) was treated with 35.7ml KOH(10%) and stirred at 250RPM at 29°C for 20 min,
5. Allowed to settle for 16 hrs,
6. Two layers were separated by decantation, top layer (oil) and bottom layer (oryzanol enriched aqueous layer)
7. Bottom layer was acidified with 2 N HCI(26.4ml) and washed once with water
8. Dried under vacuum at 80°C for 10-12 hrs to get a natural oryzanol enriched rice bran oil ready for use in foods having a desirable oryzanol content greater than five times the starting rice bran oil
9. Top layer was washed with hot water
10. The oil layer was dried under vacuum at 80°C for 1-2 hrs to get original cooking oil having oryzanol content of 0.3-0.4%
11 .Aqueous layer was sun dried for a week to get potassium chloride (salt)

Table-1: Preparation of oryzanol enriched rice bran oil (OZ-RBO)
(Table Removed)
Table-2: Characteristics of the oryzanol enriched rice bran oil
(Table Removed)
Table-3: Recovery of rice bran oil after processing
(Table Removed)
Table-4: Characteristics of oil (after removing Oryzanol enriched fraction)
(Table Removed)
Advantages:
The advantages of the invention are that a rice bran oil enriched with oryzanol to the extent of greater than five fold the content present in the starting rice bran oil has been prepared in the invention without using any organic solvent and the oryzanol enriched rice bran oil can be straight away u»ed for application in foods at desired levels. So far only oryzanol concentrates were available having 30-40% oryzanol along with the remaining 60-70% fatty material which may contain toxic solvent residues, and oxidized fatty matter arising out of long storage of soapstocks or drastic treatments during molecular distillation and other procedures adopted for the preparation of oryzanol concentrates, but the oryzanol enriched rice bran oil prepared in this invention contains good quality rice bran oil free of oxidized fatty material. The other advantages of the process for industrial adoption are that there is no solvent usage for extraction of oryzanol into oil phase, and the main product and byproducts can be re-used for edible/industrial purposes and the process can be adopted in the industry with minimal additional machinery.

We claim:
1. A process for the preparation of Oryzanol Enriched Rice Bran Oil" which comprises the steps of:
a. subjecting rice bran oil to alkali treatment with a standard alkali
solution equivalent to the real free fatty acids content, selected from
sodium hydroxide, sodium carbonate, potassium hydroxide, and
calcium hydroxide
b. mixing at 250 - 450 RPM for a period of 10 - 30 minutes,
c. settling for a period of 1 hr to 48hrs for separation of bottom soap
layer and top oil layer,
d. separating top oil layer containing oryzanol and bottom layer
containing soap and rejecting the bottom soap layer as soap stock,
e. treating the top oil layer at the controlled alkali treatment with a
lower strength stronger alkali solution selected from sodium
hydroxide and potassium hydroxide at a rate of 5-6%
f. mixing at 250 - 450 RPM for a period of 10 - 30 minutes,
g. settling for a period of 1 hr to 48hrs for separation of bottom layer
and top oil layer,
h. separating top layer and the bottom layer,
i. treating of bottom layers with dilute acid, selected from hydrochloric, sulphuric, acetic or citric acids,
j. washing the excess acid with water followed by drying under vacuum to obtain the oryzanol enriched rice bran oil.
k. treating top layer (oil) with dilute acid and washing with hot water followed by drying under vacuum to recover the spent oil for edible purposes.
2. A process as claimed in claim 1, above, the alkali used has a strength of minimum of 10%,
3. A process as claimed in claims 1 & 2, the mixing temperature is ranging from 15degree centigrade to 60 degree centigrade,
4. As claimed in claims 1 to 3, above, the starting oil is crude, degummed or dewaxed or physically refined rice bran oils,
5. As claimed in claims 1 to 4, above, the oryzanol enriched rice bran oil has a oryzanol content of 3-15%, having the 24-methylene cycloartanyl ferulate content of 10-60%,
6. A process for the preparation of Oryzanol Enriched Rice Bran Oil substantially as herein described with reference to examples accompanying this specification.

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Patent Number

270186

Indian Patent Application Number

740/DEL/2005

PG Journal Number

49/2015

Publication Date

04-Dec-2015

Grant Date

30-Nov-2015

Date of Filing

31-Mar-2005

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH

Applicant Address

ANUSANDHAN BHAWAN, RAFI MARG, NEW DELHI-110 001, INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	SAKINA KHATOON	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE-570 020, KARNATAKA, INDIA
2	A.G. GOPALA KRISHNA	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE-570 020, KARNATAKA, INDIA

PCT International Classification Number

C07J9/00; C11B13/02; C11B13/00; A61K35/7

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA