Title of Invention

"A PROCESS FOR THE PREPARATION OF ORYZANOL FROM THE RICE BRAN OIL-SOAP-STOCK"

Abstract

An improved process for preparation of oryzanol from rice bran oil soap-stock, which comprises partitioning the soap-stock with water: polar solvent : non polar : solvent at a ratio 0.6:1.5:15 ( v/v/v/ ), optionally water contains dilute acid such as hydrochloric acid at a ratio 2:1, extracting non-polar phase by dilute alkali, separating aqueous phase followed by neutralizing with acid such as defined herein, extracting obtained aqueous phase repeatedly with hexane followed by water, recovering the oryzanol by conventional solvent extraction method, said process is characterized in using partition technique consisting of aqueous polar-non polar solvent.

Full Text

The present invention relates to a Process for Preparation of Oryzanol from the Rice Bran Oil Soap-stock. Oryzanol is the name given to the class of phenolic compounds naturally occurring in rice bran oil. Principally, these are ferulic acid esters (ferulic acid esterified to cycloartenol, 24-methylene cycloartanol, beta-sitosterol and campesterol). Oryzanol is known to have a number of biological effects such as hypocholesterolemic, anti-itching, anti-dandruff, anti-aging and anti-oxidative effects as demonstrated in experimental animals. Oryzanol has also found application as a food additive for imparting antioxidant, preservative and antibiotic effects. Therefore, it is important to recover oryzanol and use it for food/pharmaceutical/cosmetic applications in the country. Rice bran oil contains 1.5-2.9% of oryzanol and during refining, about 90% of the oryzanol goes into the soap-stock as a refinery waste. Therefore, it is proposed to recover oryzanol from the soap-stock of rice bran oil. With the production of about 2 lakh tonnes of refined rice bran oil, there is a possibility of recovering as much as 4,000 tonnes of oryzanol. The literature on oryzanol started from 1954 when Kaneko and Tsuchia (Chemical Abstracts Vol.,49, Abstract No.4897b, Abstract Year. 1955) reported for the first time a phenolic substance in rice bran and rice germ oils. So far we have 9 patents out of which 5 deal with oryzanol preparation and the rest deal with products prepared from it. During the last 25 years, about 28 references have appeared in the literature which mainly deal with the biological and technological aspects viz., hypocholesterolemic effect, estrogen promoting effect, antiaging effect, antioxidant effect, and many other pharmacological, toxicological, cosmetic and food uses of oryzanol. The patent search has revealed that there are 36 patents dealing with the application of oryzanol, ferulic acid esters and rice bran oil in various products. Also there is no recent patent on the extraction and preparation of oryzanol concentrate from rice bran oil soap-stock. The early literature on oryzanol extraction from rice bran oil and its soap-stock has been covered under Japanese (i.Patent Number:4895, Patent Year:1957; ii.Patent Number:4297, Patent Year:1959; iii.Patent Number:13649, Patent Year:1960; iv.Patent Number:6812730, Patent Year:1968; v.Patent Number:76123811, Patent Year:1976) and German (Patent Number:1301002, Patent Year:1969) patents. The preparation of products with oryzanol as one of the ingredients is also covered under Japanese patents (i.Patent Number:7032078, Patent Year:1970; ii.Patent Number:7899339, Patent Year:1978; iii.Patent Number:7936306, Patent Year:1979). The pharmacological, toxicological, cosmetic and food uses of oryzanol have been well documented in the literature. However, no process is available for the preparation of oryzanol from rice bran oil soap-stock (which is carried away in the soap-stock as a refinery waste) although it has been prepared for experimental purposes by making use of patent information/procedures. As more number of industries are processing edible grade oil from rice bran and want it to be promoted as a cooking oil, entrepreneurs are looking forward to recover the value-added by-products from the refinery wastes of the oil to bring down the cost of production as well as promotion of products such as oryzanol to counter the effects of liberalisation resulting in flooding of Indian market with similar products of other countries such as Japanese products. Although oryzanol has been produced commercially in a rice growing country like Japan, it has not attracted the entrepreneurs' attention in India due to problems of processing of rice bran oil in India. However, with increase in production of edible grade rice bran oil, improvement of quality of the oil followed by recovery of value-added by-products is of primary concern for the Indian entrepreneurs. With the better understanding of the cholesterol-lowering effects of rice bran oil, large amounts of rice bran processing units are being set up in advanced countries such as U.S.A. Japan is already producing oryzanol [process and products covered under five Japanese patents (i.Patent Number:4895, Patent Year:1957; ii.Patent Number:4297, Patent Year:1959; iii.Patent Number:13649, Patent Year:1960; iv.Patent Number:6812730, Patent Year:1968; v.Patent Number:76123811, Patent Year:1976) and one German patent(Patent Number:1301002, Patent Year:1969)] and is being used in various applications. The applications include cosmetics (Japanese Patent Number:7032078, Patent Year:1970) medicated products such as anti-itching and anti-dandruff shampoos (Japanese Patent Number:7936306, Patent Year:1979) and as dentifrices (Japanese Patent Number:7899339, Patent Year:1978). Therefore, India can produce as on today, about 4000 tons of oryzanol valued at Rs.400 crores(@ Rs.lOOO/kg of approx. cost of oryzanol) which is equivalent to half the selling price of crude rice bran oil itself (about 5 lakh tons of crude rice bran oil production valued at Rs.l5/kg), therefore considered a high-value byproduct. Oryzanol so produced could capture the export market in the beginning and later on could meet the internal demand of the country in the food, pharmaceutical and cosmetic sectors. The procedure adopted was a modification of the method quoted and used by Seetharamaiah and Prabhakar[G.S.Seetharamaiah and J.V.Prabhakar, Oryzanol conent of Indian rice bran oil and its Extraction from soap-stock, J. Food Science & Technology, Vol.23, pages,270-273, (1986)]. The procedure adopted has been taken from the Japanese patent of Tsuchiya and Kato (Patent Number:4297 and Patent Year:1959) wherein the method of preparation makes use of extraction of oryzanol from an aqueous suspension of the soapstock of rice bran oil with diethyl ether in the first step followed by the second step of dissolution of the oryzanol fraction in an alkali solution, followed by recovery of oryzanol with diethyl ether from the alkali solution after neutralization to get the oryzanol concentrate. In the method used by Seetharamiah and Prabhakar, the pH of the soapstock of rice bran oil was adjusted before extraction with diethyl ether in the first step. Later, the oryzanol concentrate of approximately 16% purity has been purified by alumina column chromatography to achieve a purity of 51% oryzanol content in the purified material. The draw backs in these procedures is that the volume of aqueous suspension is so high (20 times the volume of raw material and solvents used) that problems of emulsification and incomplete extraction exist. Also, the use of a highly inflammable and low boiling solvent, such as diethyl ether for extraction makes the process more accident prone and hazardous to the workers. In spite of these, the purity of the product achieved is not more than 17%. Further purification steps are necessary to upgrade the product which is expensive and time consuming. Therefore, in the present invention, we have addressed these problems of emulsion formation and accident prone low boiling solvent usage for extraction and have come out with a better purity product by the solvent partition method alone without resorting to column chromatographic purification. The oryzanol purity achieved in the present invention is 65%. The process is also quicker compared to the existing methods of extraction. The main object of the present invention is to provide a process for the preparation of oryzanol concentrate from the soap-stock of rice bran oil which obviates the drawbacks as detailed above. Another object of the present invention is to modify the oryzanol in the form of concentrate to be used in various preparations such as in the food, for imparting instantizing property to the ready mixes. Yet another objective of the process of this invention, is to make use of combination of immiscible polar and non-polar solvent for extraction of oryzanol in place of polar solvent used in hitherto known process. Further, two step neutralization with acid also helps in getting high purity of the product (50-65%) using simple techniques avoiding column chromatography. Yet another object of the present invention is to explore the possibilities of use of the oryzanol concentrate in pharmaceutical preparations such as for lowering cholesterol. In addition to the above, another object of the present invention is to use the orvzftnoi concentrate in cosmetic preparations for Accordingly the present invention provides an improved process for preparation of oryzanol from rice bran oil soap-stock , which comprises partitioning the soap-stock with water: polar solvent : non polar solvent at a ratio 0.6: 1.5 : 15 ( v/v/v) , optionally water contains dilute acid such as hydrochloric acid at a ratio 2:1 , extracting non-polar phase by dilute alkali , separating aqueous phase followed by neutralizing with acid such as defined herein , extracting obtained aqueous phase repeatedly with hexane followed by water , recovering the oryzanol by conventional solvent extraction method , the said process is characterized in using partition technique consisting of aqueous polar-non polar solvent . The soap-stock was treated with water/ethanol/hexane solvent system in the ratio of water:ethanol:hexane(water containing a dilute acid in the ratio of 2:1) (0.6:1.5:15, v/v/v) to extract the fatty layer in the first instance followed by recovery of oryzanol from that layer using alkali treatment in the ratio solvent extract:alkali (15:2 v/v) in single or 3-5 repeated batches using same solvent and fresh alkali followed by subsequent neutralization with a dilute acid (mineral or low molecular weight organic acid of chainlength C1-C3 and recovery of oryzanol using the same organic solvent in fresh batches/or in repeated extraction procedure developed in the laboratory. The results are included in Table 1. The recovered product has a purity of 15.4-65%. In another embodiment off the present invention, the immiscible non-polar and polar-solvents used are selected from aliphatic C5 to C8 Carbon chain length containing hydrocarbon solvents as a non-polar solvent and of aliphatic CI to C4 carbon chain length hydroxyl group or oxygen containing compound as polar solvents selected from diethyl ether, methanol, ethanol, propanol, isopropanol or mixture thereof. In still another embodiment of the process invented as detailed earlier, the alkali used is an inorganic alkaline/alkaline earth metal hydroxide of strength between 0.1 normal to 10 normal solutions. In a still another embodiment of this invention, the acid used for neutralization is either an inorganic or an organic acid of basicity 1 or 2 and could be made of aliphatic carbon chain length of the range CI to C3 such as formic, acetic and propionic acids or sulphuric, hydrochloric or nitric acids and prepared as an aqueous solution of strength between 0.1 to 10 normal solutions. In still another embodiment of this invention, the reactions are effected at a temperature between 20 and 50°C. The new findings are that i) selection of a nonpolar solvent such as hexane for extraction of oryzanol and ii) use of a pH of 8.2 for the extraction of a better purity oryzanol using hexane as the extracting solvent from the alkaline solution of oryzanol. iii) The new findings are that some interfering materials are found in rice bran oil soap-stock presumably, the degradation products of phospholipids which have absorption at 314 nm and therefore oryzanol content determination to be confirmed by HPLC analysis. The oryzanol concentrate prepared had the following characteristics: Colour and appearance: Viscous dark to pale yellow pasty mass to dark brown crystalline powder to amorphous pale yellow powder with a reddish orange tinge. Melting Point of the powder: approx. 85-105°C max :314nm, E1% lcm at 314nm in hexane:53.84-233.29 Chemical composition by HPLC: i. Cycloartenyl ferulate:16.6-28.8% ii. 24-Methylene Cycloartanyl ferulate:35.4-42.7% iii. Campesteryl ferulate:18.9-25.2% iv. Beta-sitosteryl ferulate:9.6-16.8% The commercial products of rice bran oil industry had a oryzanol content of 1.8-2.0% for crude oil, 0.19-0.22% in refined oil, 2.21-6.71% in the soapstock. The commercial soapstocks obtained from two manufacturers showed a oryzanol content of 6.3-6.9%. By using a combination of non-polar and polar solvents for the extraction of soapstock followed by alkali neutralization the oryzanol content could be enriched to 15%-65%. Four batches of the commercially produced soapstocks obtained from two entrepreneurs were subjected to solvent partition technique using polar and non-polar solvent combination followed by alkali neutralization to get a concentrate of 15-65% oryzanol content starting from commercial soapstock (oryzanol content of 2.82%) with an yield varying from 22-35% (for 38-65% purity) to 76% (for 15-27% purity). The process of the present invention is illustrated by the following examples, however, it should not limit the scope of this invention. Example 1 To 10 grams of soap-stock was added 6 millilitres of water and 15 millilitres of ethanol and mixed with 150 millilitres of hexane in an Erlenmeyer flask of 250 mL capacity. After 5 min. of mixing, the hexane phase was separated and extracted with 5 millilitres of potassium hydroxide solution(lN) and separated the aqueous phase. The aqueous phase was neutralised with hydrochloric acid solution (IN) completely and extracted with hexane (25 millilitres X 3) and the extract pooled, washed once with water, desolventized to get the oryzanol concentrate of 11.63% purity (Table 1). Example 2 To 10 grams of soap-stock taken in 10 batches was added 6 millilitres of water and 15 millilitres of ethanol and mixed with 150 millilitres of hexane as described in example 1 above. The hexane phase separated and treated with 5 millilitres of potassium hydroxide solution and the aqueous phase collected separately and preserved. The hexane phase is now treated with a fresh batch of 10 grams of soap-stock and solvent mixture and the process repeated on 9 batches. The aqueous alkali phase obtained from all the 10 batches were pooled and treated with hydrochloric acid as in example 1 and extracted with hexane in one step using 250 millilitres X 3 of the solvent which after desolventization yields the oryzanol concentrate having 15.4%-24.7% oryzanol(Table 1). Similarly the purity of the product prepared could be improved by two stage extraction but with low yield (Table 1) without affecting the total recovery. Example 3 To 56 grams of soap-stock was treated with 33.6 millilitres of water containing 11.2 millilitres of hydrochloric acid (1N) and 84 millilitres of ethanol and mixed with 810 millilitres of hexane in a beaker of 5 litres capacity for 5 minutes at 2 7°C. Then the hexane phase separated and the mother liquor preserved for subsequent 2nd, 3rd, 4th and 5th extractions with same solvent. The separated hexane phase is now treated with 28 millilitres of the alkali solution (mentioned in example 1) and separated the aqueous phase. The aqueous phase was collected similarly from the hexane phase after the 2nd, 3rd, 4th and 5th extractions on the mother liquor and all the five extractions pooled. Then the alkali solution was adjusted to pH 8.2 with the acid (mentioned in example 1) and extracted with hexane (140 ml X 3) and the extracts pooled, washed once with water and desolventized to get oryzanol of 53.4% purity. Then, the mother liquor alkali solution from the above extraction is treated with additional amounts of the acid to get a pH of about 3 followed by a second extraction with hexane (140 ml X 3) and the extracts pooled, washed once with water and desolventized in the usual way to get oryzanol concentrate of 15-25% purity. The total recovery of oryzanol from the method achieved was 80-88.1% (Table 1). Using the above procedure, a batch of 280 grams of soap-stock was subjected to extraction and the product prepared had a purity of 65% oryzanol(Table 1). Table 1 Extraction of oryzanol using same solvent for repeated extractions (Table Removed) The main advantages of the present invention are i. the solvent(s) allowed in the food industry is(are) used in the process. ii. a low boiling and highly inflammable solvent viz., diethyl ether used by earlier workers has been replaced with hexane which has a high boiling point and does not develop peroxides. iii. to prevent emulsion formation, the extraction is carried out with a solvent system making use of minimum amount of water and therefore absolute control over extraction has been achieved, iv. the solvent ethanol (or rectified spirit) used in the present invention is allowed in the food industry unlike methanol used in other processes/methods which is hazardous. v. The oil industry can make use of the fat extraction solvent viz., commercial hexane for the purpose without resorting to purchase of an additional solvent, viz., diethyl ether, vi. The solvent used in the first step can be repeatedly used in the same step without the need for use of fresh solvent, thereby the process is economical and can be made continuous, vii. Because of no emulsion formation, the solvent phase separation is very quick and therefore, it is a quicker process, viii. The adjustment of pH of 8.2 to get a high purity oryzanol using hexane as the extracting solvent in the second step of extraction is new and novel unlike, the procedure of Seetharamaiah and Prabhakar wherein a pH of 9-9.5 has been suggested for total extraction of oryzanol in the first step and using diethyl ether as the solvent from an aqueous phase, ix. Fatty acids and lecithin and or wax are obtained as byproducts of the process. We claim: 1. An improved process for preparation of oryzanol from rice bran oil soap-stock , which comprises partitioning the soap-stock with water: polar solvent: non polar solvent at a ratio 0.6: 1.5 : 15 ( v/v/v), optionally water contains dilute acid such as hydrochloric acid at a ratio 2:1 , extracting non-polar phase by dilute alkali , separating aqueous phase followed by neutralizing with acid such as defined herein , extracting obtained aqueous phase repeatedly with hexane followed by water , recovering the oryzanol by conventional solvent extraction method, said process is characterized in using partition technique consisting of aqueous polar-non polar solvent . 2. An improved process as claimed in claim 1 wherein the non-polar and polar solvents used are selected from aliphatic C 5 to C 8 carbon chain length containing hydrocarbon solvents, preferably hexane as a non polar solvent and of aliphatic C1 to C4 carbon chain length hydroxyl group or oxygen containing compound as polar solvents such as diethyl ether , methanol, ethanol, propanol, isopropanol or mixture thereof. 3. An improved process as claimed in claim 1 and 2 wherein the alkali used is an inorganic alkaline /alkaline earth metal hydroxide such as potassium hydroxide of strength ranging 0.1 to 10 (N). 4. An improved process as claimed in claim 1 and 3 wherein acid used for neutralization is selected from inorganic acid such as hydrochloric acid , nitric acid or sulphuric acid at a strength ranging 0.1 to 10 (N) or organic acid of basicity 1 or 2 of aliphatic carbon chain length of the range C1to C3 such as formic acid , acetic acid and propionic acids. 5. An improved process for preparation of oryzanol from rice bran oil soap-stock substantially as herein defined with reference to the examples .

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