Title of Invention

AN IMPROVED PROCESS FOR THE PREPARATION OF THERMOSTABLE EMULSIFIER

Abstract

An improved process for the preparation of thermostable emulsifier using yarrovvia lipolytica sp., which comprises growing the said species in a fermentation medium prepared in sea water containing a carbon, nitrogen and phosphate source, for 4 to 7 days at a temperature ranging between 28 to 32°C in sterile conditions, harvesting the medium, separating the biomass using conventional methods, concentrating the liquid containing the emulsifier by conventional methods, separating the emulsifier from this concentrated liquid by isoelectric focussing to obtain the emulsifier.

Full Text

This invention relates to an improved process for the preparation of thermostable emulsifier. More particularly it relates to the preparation of the said emulsifier using Yarrowia lipolytica sp. The thermostable emulsifier prepared according to the process for the present invention is active in a wide range of pH that is 2.0 - 10.0 and could be prepared by conventional fermentation using Y. lipolytica in a medium containing alkane as the sole carbon source. Microbial emulsifiers have applications in enhanced oil recovery operations, oil spill management, in textiles, Pharmaceuticals, cosmetics and food industry. Effectivity of microbial emulsifiers has also been explored as an alternative method to disperse pollution created by routine shipping operations and major oil spills in the marine ecosystem [ Fiechter A. (1992) Trends in Biotechnology, 10:208-217]. Marine yeasts have not been studied extensively for the production of surfactants which may have unique properties. In the prior art, microbial emulsifiers have been isolated from various sources. However, the general procedures used to obtain these emulsifiers in the refined form are tedious and the final yield is low. Only partially refined preparations are reported from a few microbial sources for example, a bioemulsifier from Candida lipolytica reported by Cirigliano and Carman has been isolated by using atleast 6 steps involving the use of hazardous organic solvents like chloroform-methanol (2:1) mixture. Moreover, the yield of the bioemulsifier is barely 30% (Appl. Environ. Microbiol. 48:747-750, 1984). In another procedure for the separation of a biosurfactant (bioemulsifier) from a different strain of Candida lipolytica, the steps involving the use of a foaming device and organic solvents has been reported without ascertaining the purity of the product. (Parei 1 leux, A. Eur. J. Appl. Microbiol. Biotechnol . 8: 91-101, 1979). Shohan et a7 used 5 different steps to obtain purified bioemulsifier from Acineto-bacter calcoaceticus RAG-1. This long procedure also involves the use of organic solvents (Shoham,Y., Rosenberg, M. and Rosenberg, E. Appl. Environ. Microbiol. 46 : 573-579, 1983). The inventors of the present invention have, during their course of research, isolated a yeast species from oil contaminated sea water near Mumbai ( Maharashtra, India), by enrichment with 2 % crude oil. It was identified as Yarrowia lipolytica on the basis of conventional tests and was found to produce a useful and thermostable emulsifier. Using the same culture the inventors have invented a simplified, eco-friendly and rapid procedure for the preparation of a salt-tolerant, thermostable emulsifier with high yields. The object of the present invention is to provide an improved process for the preparation of thermostable emulsifier using Yarrowia lipolytica sp, Another object is to provide a rapid process for the isolation of the emulsifier for its possible use because isolation of emulsi-fiers from fermentation broths is generally a tedious process. Accordingly, the present invention provides an improved process for the preparation of thermostable emulsifier using Yarrowia lipolytica sp., which comprises growing the said species in a fermentation medium prepared in sea water or artificial sea water containing a carbon, nitrogen and phosphate source, for 4 to 7 days at a temperature ranging between 28'C to 32 °C in sterile conditions, harvesting the medium, separating the biomass using conventional methods, concentrating the liquid containing the emulsifier by conventional methods, separating the emulsifier from this concentrated liquid by isoelectric focussing to obtain the emulsifier. In one of the embodiments of the present invention the carbon source used in the fermentation medium is selected from dodecane, tertadecane, hexadecane or octadecane. In another embodiment of the invention the nitrogen source is selected from ammonium salts, preferably ammonium sulphate. In yet another embodiment of the present invention the phosphate source in the "fermentation medium is selected from di-sodium or di-potassium hydrogen phosphates. In a feature of the invention the concentration of the liquid containing the emulsifier could be carried out by conventional methods like ultrafi1tration, alcohol precipitation, or acetone preci pi tation. In a feature of the invention the organism, Yarrowia lipolytica is cultivated on artificial sea water containing 1 % hexadecane, 0.001 % dipotassium hydrogen phosphate and 0.5 % ammonium sulphate. The cultivation was carried out for 6 days. After harvesting the crude broth and filtering through YM-10 membrane the concentrate was subjected to isoelectric focusing in a minis-cale modified unit. Description of this unit is as follows: It consists of two vertical glass tubes ( 8 X 430 mm.) joined with a polyester tubing. A small cock attached to the right arm of the U-tube facilitates easy collection of the emulsifier without disturbing pH gradient. Following solutions were prepared: Light density solution: Water, 4.5 ml; ampholine carrier electrolytes (pH range 3.5 - 10.0), 0.2 ml; and the sample 0.5 ml. Heavy density solution : Glycerol, 3.0 ml; water, 1.7 ml; carrier electrolytes, 0.2 ml and the concentrated emulsifier sample containing approximately 15 units, 0.5 ml. Separation solution: Glycerol, 3.0 ml and water, 2.0 ml. Cathode solution: 1 M Na OH, 2.5 ml and water, 7.5 ml. Anode solution : 1 M o-phosphoric acid, 4.0 ml and sucrose 15.0 g, diluted to 25 ml with water. di-potassiurn hydrogen phosphates. In a feature of the invention the concentration of the liquid containing the emulsifier could be carried out by conventional methods like ultrafi1tration, alcohol precipitation, or acetone precipitation. In a feature of the invention the organism, Yarrowia lipolytica is cultivated on artificial sea water containing 1 % hexadecane, 0.001 % dipot-assium hydrogen phosphate and 0.5 % ammonium sulphate. The cultivation was carried out for 6 days. After harvesting the crude broth and filtering through YM-10 membrane the concentrate was subjected to isoelectric focusing in a minis-cale modified unit. Description of this unit is as follows: It consists of two vertical glass tubes ( 8 X 430 mm.) joined with a polyester tubing. A small cock attached to the right arm of the U-tube facilitates easy collection of the emulsifier without disturbing pH gradient. Following solutions were prepared: Light density solution: Water, 4.5 ml; ampholine carrier electrolytes (pH range 3.5 - 10.0), 0.2 ml; and the sample 0.5 ml. Heavy density solution : Glycerol, 3.0 ml; water, 1.7 ml; carrier electrolytes, 0.2 ml and the concentrated emulsifier sample containing approximately 15 units, 0.5 ml. Separation solution: Glycerol, 3.0 ml and water, 2.0 ml. Cathode solution: 1 M Na OH, 2.5 ml and water, 7.5 ml. Anode solution : 1 M o-phosphoric acid, 4.0 ml and sucrose 15.0 g, diluted to 25 ml with water 0.5%) before cultivation in submerged culture using following sterile medium: Ammonium sulphate, 0.5 %, di-potassium hydrogen phosphate 0.001 % and octadecane 1 %, in Arabian sea water. The fermentation was carried out for 6 days and the broth was harvested, and centrifuged. The liquid containing the emulsifier was concentrated by ultrafi1tration. The concentrate was subjected to isoelectric focussing for purification. The concentrate is further lyophilized to obtain the emulsifier in powder form. EXAMPLE 2 The yeast strain Y. lipolytica was cultivated in submerged culture using following sterile medium: Ammonium nitrate, 0.5 %, di-sodium hydrogen phosphate 0.001 % and hexadecane 1%, in Arabian sea water. The fermentation was carried out for 7 days and the broth was harvested, and centrifuged. The liquid containing the emulsifier was concentrated by alcohol precipitation. The concentrate was subjected to isoelectric focussing for purification. The concentrate is further lyophilized to obtain the emulsifier in powder form. EXAMPLE 3 The yeast strain Y. lipolytica was cultivated in submerged culture using following sterile medium: Ammonium chloride, 0.5 %, di-potassium hydrogen phosphate 0.001 % and tetradecane 1%, in artificial sea "water. The fermentation was carried out for 6 days and the broth was harvested, and centrifuged. The liquid containing the emulsifier was concentrated by ultrafi1tration. The concentrate was subjected to isoelectric focussing for purification. The concentrate is further lyophilized to obtain the emulsifier in powder form. The major advantages of this inventions are as follows: 1. The procedure involving isoelectric focusing is most rapid and simple for the separation of emulsifier from the cell free extract. This technique is used first time for the isolation of the microbial emulsifier. Since it is rapid, many microbial emulsifiers can be obtained in the refined form to test their characteristics. 2. The organism, namely Yarrowia lipolytica is non- pathogenic in nature and therefore facilitates its use in various fields. 3. The emulsifier in the present invention is highly stable to adverse environmental conditions and therefore it can find many applications in future. We claim : 1 . An improved process for the preparation of thermostable emulsifier using Yarrowia lipolytica sp., which comprises growing the said species in a fermentation medium prepared in sea water containing a carbon, nitrogen and phosphate source, for 4 to 7 days at a temperature ranging between 28 to 32*C in sterile conditions, harvesting the medium, separating the biomass using conventional methods, concentrating the liquid containing the emulsifier by conventional methods, separating the emulsifier from this concentrated liquid by isoelectric focussing to obtain the emulsifier. 2. A process as claimed in claim 1 wherein the carbon source used in the fermentation medium is selected from dodecane, tertadecane, hexadecane or octadecane. 3. A process as claimed in claim 1 wherein the nitrogen source is selected from ammonium salts, preferably ammonium sulphate 4. A process as claimed in claim 1 wherein the phosphate source in the fermentation medium is selected from di-sodium or di- potassium hydrogen phosphates. 5. An improved process for the preparation of thermostable emulsifier using Yarrowia lipolytica sp., as substantially described hereinbefore with reference to examples.

Documents:

253-del-1997-abstract.pdf

253-del-1997-claims.pdf

253-del-1997-complete specification (granted).pdf

253-del-1997-correspondence-others.pdf

253-del-1997-correspondence-po.pdf

253-del-1997-description (complete).pdf

253-del-1997-form-1.pdf

253-del-1997-form-19.pdf

253-del-1997-form-2.pdf