Title of Invention

"A PROCESS FOR THE PREPARATION OF INACTIVATED PLANT LIPASES."

Abstract

The present invention relates to a process for the preparation of inactivated plant lipases .More particularly, the present invention relates to a process for the preparation of inactivated wheat germ and rice bran lipases using selected biomolecules. Inactivated lipases are prepared by incubating the active lipase enzyme and a ligand selected from chlorogenic acid and caffeic acid and an activator and a substrate to produce the inactivated lipases.

Full Text

Field of invention The present invention relates to a process for the preparation of inactivated plant lipases . More particularly, the present invention relates to a process for the preparation of inactivated wheat germ and rice bran lipases using selected biomolecules. Background of the invention The rice grain contains 2-3% fat, most of which is concentrated in the embryo or germ and in the outer seed layers. Milling of rice separates the germ and bran layers from the endosperm and concentrates the fat in the residue commonly known as "bran" which contains 10-26% oil. Although rice bran has considerable potential as a contributor to world oil supply, it is seldom considered in the list of edible oil raw material sources. Rice bran oil is seen as a superior oil, rich in vitamins and low in ingredients responsible for cholesterol. Rice bran oil can play a significant role in augmenting India's domestic vegetable oil supply. Despite being a leader in rice production, India still has a long way to go in the recovery of good quality rice bran to produce food-grade oil. If properly harnessed, rice bran - an important byproduct of the rice milling industry - can be used to augment the domestic edible oil supply. The prime draw back of rice bran as a source of edible oil is associated with its deterioration in storage, principally, as a result of hydrolysis of oil by lipolytic enzymes present in the bran. The lipolytic activity is observed as soon as the bran is removed from the rice and lipolysis continues with prolonged storage , yielding high levels of free fatty acids in the oil, which becomes unsuitable for processing to an edible oil. In India, time delay between milling and oil extraction is responsible for increases of the free fatty acid content from 2.5-3.0% in the freshly milled bran to beyond 10% which results in high refining losses. Therefore, a number of approaches have been employed to arrest the lipolysis in the bran and the process termed "stabilization of rice bran" uses cumbersome and very expensive methods. Several designs such as chemical, mechanical and thermal, have been attempted. The investigation of the effect of heating or drying and the effect of different relative humidities on the storage of rice bran have shown that rice bran can be stored for periods of at least four months without excessive increase in the content of free fatty acids. An increase in the moisture content of predried bran causes a rapid increase in the free fatty acid content of the oil. Another approach to combat this problem is sterilization of bran at elevated temperatures and moistures to retard the action of the enzyme sufficiently to permit the storage of the bran with no rise in free fatty acid for reasonable lengths of time . A practical solution to the stabilization of rice bran is to store the material at low temperature. Other methods to stabilize rice bran like gamma irradiation or chemicals or microwave heating generally have not been successful. Studies also have shown that methanol at 2% v/w and concentrated HCI spray could not arrest the release of free fatty acid from the rice bran oil in comparison to other organic solvents and mineral acids. In addition, the methanol or HCI in the presence of metal ions did not influence the oil content of rice bran during storage. There are also reports that Fe3+ and Ni2+ could be effectively used to arrest the release of free fatty acids in rice bran and thus contribute to improving the edible oil quality of rice bran oil. However, the feasibility of using metal ion and/or solvent-treated rice bran in the processing of food for human consumption is rather difficult in the absence of any metabolic study. Therefore, many a times, enzymes such as lipase which are undesirable in food systems need to be inactivated and hence the improved methods of stabilization of rice bran for the production of food-grade rice bran oil. In the case of wheat, the lipase activity is found to be located in the bran component of the grain(75-80%) and significant activity was also present in the germ(20-25%) . The presence of lipase activity is of both economic and nutritional importance due to its role in the oxidative rancidity. The major cause of this deterioration is thought to be due to the degradation of cereal lipids. In wheat, this appears to be initiated by the lipase-catalysed hydrolysis of triacylglycerols to unesterified fattyacids with increasing storage time. Lipases ( triacylglycerol acyl hydrolases, EC 3.1.1.3 ) constitutes one of the structurally less characterized families of hydrolases. The natural substrates of lipases are triacylglycerols having very low solubility in water. They hydrolyze the ester bonds in tri, di and monoacylglycerols, although some are known to degrade fairly broad range of compounds containing an ester linkage. Lipase activity has been identified in a wide range of oilseeds. In oilseeds, containing 20-50% lipids(mainly triacylglycerols) as storage material, it is obvious that lipase action is essential in germination when reserve(insoluble) triacylglycerols are converted to soluble sugars which can be transported to the growing embryos to supply carbon and energy. Oil seed lipases from diverse plant species exhibit differences in their substrates, pH optimum ,and reactivity towards sulfhydryl reagents, hydrophobicity and subcellular location. Seed lipase from a certain plant species is relatively specific for the native triacylglycerols or triacylglycerol containing the major fatty acids of the storage triacylglycerols of the same species.With the exception of castorbean lipase, the lipase activities are very minimal or absent in ungerminated seeds and are found to increase after germination. In rapeseed and mustard seed, the lipase is more active in germinated seed as compared to ungerminated seed. True lipases, which attack the fatty acyl linkage of water-insoluble triacylglycerols, are known to occur in oilseeds and grains. In postgerminative growth, the oil reserve is rapidly mobilized to provide energy and skeletons for the growth of the embryo. The triacylglycerols are localized in subcellular glyoxysomal membrane and contains an acyl hydrolyze/true lipase. In seeds where the lipase is found in subcellular compartments other than the lipid bodies, the enzymes still have to come in contact with membrane of the lipid bodies during catalysis. Fatty acids liberated due to the action of lipolytic enzymes are water-insoluble and its removal from the lipid bodies is of prime importance and essential for the continuation of the lipdlysis in the plant. There are several possible mechanisms for the transport of fatty acids. The important among them is the membrane flow mechanism. The fatty acids are released by the action of lipases on the lipid body membrane and flow along the membrane by a simple diffusion through the contact junction of the two organelles due to a concentration gradient to the membrane of the glyoxysome, similar to the one proposed for the mamalian systems. The physiological role of the glyoxisomal lipase in lipolysis has not been substantiated but still there exists a possibility that it is involved in the transport of fatty acids. Lipase activity plays an important role in the germination and postgermination growth of oilseeds where stored triacylglycerols are used in the first reaction of the gluconeogenic pathway. In tissues other than leaves, alterations of membrane lipids occur in development and differentiation such as during seed maturation and germination and fruit ripening. The released free fatty acids which are the source of rancidity as in the case of rice bran , from the enzymatic reaction may undergo oxidation catalyzed by oxidases to produce volatile and nonvolatile metabolites that may be of normal nature. All lipases consists of a catalytic triad of His-Ser- Asp/Glu, reminiscent of serine proteases. The defining characteristic of lipases is the phenomenon termed " interfacial activation " which is caused by a change of its conformation resulting from adsorption. Many industrial fronts have shifted towards utilizing this enzyme for a variety of reaction of immense importance in dairy, pharmaceutical, agrochemical and industries due to its efficiency to catalyze a variety of esterification and transesterification reaction. Biomolecules, such as chlorogenic acid(CGA) ,caffeic acid(CA) and quinic acid(QA) play a significant role in many biological and metabolic processes like inhibition of lipoxygenase activity in prostaglandin metabolism, inhibition of retinoic acid 5,6- epoxidation and hydroxyl radical formation, antiviral activity and interaction with DNA. These multifunctional nature of the above selected biomolecules show that they act differently depending upon the biological processes. CGA is present in large quantities in coffee, tea and in many oil seeds in complex with proteins, and is rapidly converted to CA and QA by breakage of the ester bond between them. The CA moiety is more dominant and the principle active group in CGA which has been shown with the intestinal motility action in rats induced by these compounds. The result of this work is analyzed from the point of view of stabilization or destabilization effect on lipases. Reference may be made to O'Connor. J, Harwood, J.L . Solubilization and purification of membrane-bound lipases from wheat flour. J. Cereal. Sci. 16, 141-152(1992), wherein the effect of a variety of metal ions and enzyme inhibitors on lipase preparations were evaluated, with a view to suggesting a means of controlling their activities and showed, the addition of 100 mM NaCI to whole flour samples caused a 55% reduction in total -lipese activity, whilst treatment of flour with 100 mM NaCI before solubilization caused a complete loss in activity. However the drawback of the invention was inhibitor used at very high concentration.The influence of addition of 2, 4, 6, 8 and 10µ M of benzoic and cinnamic acids and selected phenolic acids like salicylic, p-hydroxybenzoic, gentisic, protocatechuic, vanillic, syringic, o-coumaric, p-coumaric, caffeic, ferulic, sinapic on the activity of pancreatic lipase was examined in vitro. The strongest inhibition activities were observed with caffeic, ferulic and benzoic acid, while sinapic and gentisic acids produced the lowest inhibition( Karamac M and Amarowicz R, 1996. Inhibition of pancreatic lipase by phenolic acids-Examination in vitro. Z Naturforsch., 51(11-12), 903-905). The inhibitory effect of caffeic acid analogues isolated from Salviae Miltiorrhizae Radix were examined and found that the free radical 1,1-diphenyl-2-picrylhydrazyl(DPPH) inhibited by the polymers of caffeic acid is more than caffeic acid. The strongest activity was displayed by two tetramers, lithospermic acid B and its Mg 2+ salt. The inhibitory effect of caffeic acid derivatives revealed that the o-dihydroxyl group was the most important active structure of caffeic acid derivatives for scavanging of free radicals. The saturated group connected to the aromatic ring has slightly higher inhibitory activity against the DPPH radical than an unsaturated group( Chen C.P., Yokozawa T and Chung H Y. 1999. Inhibitory effect of caffeic acid analogues isolated from Salviae Miltiorrhizae Radix against 1,1-diphenyl-2-picrylhydrazyl radical. Exp Toxicol Pathol 51(1), 59-63).The chlorogenic acid was found to be the inhibitor on the basis of UV and mass spectra and comparison with a standard sample. The inhibition of caffeic acid was also checked in 8-OH-dg formation in animal organs, an oxygen radical forming carcinogen,4-nitroquinoline-1-oxide( kasai H, Fukada, . S, Yamaizumi, Z, Sugie, S and Mori H. 2000. Action of chlorogenic acid in vegetables and fruits as an inhibitor of 8-hydroxydeoxyguanosine formation in vitro and in a Rat carcinogenis Model. Food and Chemical Toxicology, 38, 467-471).The haematin and haemoglobin-catalysed retinoic 5,6-epoxidation were inhibited by chlorogenic acid and caffeic acid suggesting that the o-hydroquinone moiety of chlorogenic acid and caffeic acid is essential to the inhibition of the epoxidation. However caffeic acid does not inhibit the formation of retinoic acid radicals but does inhibit the step of conversion of retinoic acid radical into the 5,6-epoxide( Iwahashi, H, Negoro, Y, Ikeda.A, Morishita,H and Kido.R. 1986. Inhibition of chlorogenic acid of haematin-catalysed retinoic acid 5,6-epoxidation. Biochem. J. 239, 641-646). A, chlorogenic acid, which is an ester of caffeic acid and quinic acid, inhibited the N-nitrosation of a 2,3-diaminonaphthalene by the N-nitrosating agent produced by nitrite in acidic solution. Caffeic acid was also inhibited the N-nitrosation. The observations showed that the mechanism by which chlorgenic acid inhibited N-nitrosation of 2,3 -diamino-naphthalene is due to its ability to scavenge the nitrosating agent, nitrogen sesquioxide and inhibition is effective not only in protecting against oxidative damage but also in inhibiting potentially mutagenic and carcinogenic reactions( Kono Y, Shibatg H, Kodama Y and sana Y 1995 The supressinn of the N- niuoing reactic: chlorogenic acid . Biochem. J. 312, 94 - 3). Polyphenols and their relate oounds inhibit overall fonnation oi '3- hydroperoxide ctadecadienoic acid,(HPLC-ESR) derived radicals in the reaction mixture of 13-(HPLC-ESR) in ferrous ions were examined by ESR . On the other hand the high performance quid chromatography- electron spin resonance(HPLC-ESR) and the high performance liquid chromatography- electron spin resonance -mass spectrometries(HPLC-ESR-MS) showed that caffeic acid inhibited the formation of octanoic acid radical and pentyl radical and results showed that the chelation of ferrous ion is responsible to the inhibitory effects of the polyphenols( Iwahashi, H. 2000. Some polyphenols inhibit the formation of pentyl radical and octanoic acid radical in the reaction mixture of linoleic acid hydroperoxide with ferrous ions. Biochem. J. 345, 265-273). There is lot of information available on the inhibition by biomolecules and chlorogenic acid in particular. But the information on the inhibition of plant lipases by biomolecules is meagre. Reference may be made to Grunberger, Dezider, Frenkel, Krystyna..Inhibition of cataract formation, diseases resulting from oxidative stress, and HIV replication by caffeic acid esters; US Patent 5591773 (1994) Where the method of inhibiting the formation of a cataract in an eye by contacting the eye with a caffeic acid esters and method comprising administering a pharmaceutical composition comprising the caffeic acid esters to inhibit the formation of a cataract in the eye. The caffeic acid esters prevents diseases resulting from oxidative stress. Further, caffeic acid esters may be used to treat an HIV infection when combined in a pharmaceutical composition with a substrate which inhibits HIV replication. Reference may be made to Agoro, John ,W.. Crystalline caffeic acid derivatives and compositions and method for treating snakebite; US Patent 4124724 (1978) wherein crystalline products of manufacture from the group consisting of hydroxyl and the caffeoyl group are useful as antivenom agents for hemolytic snake venoms. The caffeic acid containing hydroxyl group both in R1 and R2 positions, are provided for treatment of snakebite victims,as well as the method of using those preparations in such treatment. Reference may be made to Riemer, Jed, A. 1994. Bitterness inhibitors; US patent 5336513 wherein caffeic acid and ferulic acid were found to be the preferred bitterness inhibitors derived from cinnamic acid. A process of reducing the bitterness of consumable materials is set forth which comprises the addition of the inhibitors from about 0.001% to 0.2% by weight. Reference may be made to Isler, and Dorothea, Rehm, Walter, Widmer and Erich. 1996. Biomass lipase inhibitor useful for treating adiposity ; US Patent 5540917 where a lipase inhibitor selected from lipstatin in pure form, a biomass comprising a lipase inhibitor, and tetrahydrolipstatin were used for the treatment of adiposity and a pharmaceutical composition comprising at least one water insoluble crude fiber and at least one lipase inhibitor.Reference may be made to Mullins, John Jason Gentry. 2001. Essentially nonabsorbable lipase inhibitor derivatives, pharmaceutical compositions and methods of use thereof ; US Patent 6235305 wherein pharmaceutical comprising lipase inhibitor that have been rendered non-absorbable by linking such lipase inhibitors to a non-absorbable support and method of use therefor to treat adiposity or obesity. Reference may be made to Bremer, Klaus-Dieter, Sawlewicz, Pavel. 1997. Pharmaceutical composition comprising a glucosidase and/or amylase inhibitor, and a lipase inhibitor; US Patent 5643874 where a pharmaceutical composition containing a glucosidase and/or amylase inhibitor and a lipase inhibitor as active substances and are used as usual pharmaceutical carriers. Reference may be made to Okuda, Takuo, Yoshida, Takashi, Hatano, Tsutomu, Hashimoto, Toshitaka, Yamashita, Akiko, Shimura, Susumu, Itoh, Yoshio. 1997. Tanins and lipase inhibitors containing the same as active ingredients ; US Patent 5629338 wherein tannins and lipase inhibitors containing the same as active constituent and method for producing the same. Reference may be made to Lange, Louis,G, Spilburg, Curtis, A. 1991. Use of sulphated polysaccharides to inhibit pancreatic cholesterol esterase ; US Patent 5017565 where method comprising the composition which inhibit pancreatic cholesterol esterase and triglyceride lipase and hence, lower cholesterol and triglycerides in the blood stream. Reference may be made to Aggarwal , Bharat.B, Grunberger, Dezider. 1999. Inhibition of nuclear transcription factor NF-.Kappa.B by caffeic acid phenylester(CAPE), derivatives of CAPE, capsaicin(8-methyl-N- vanillyl-6-nonenamide) and resiniferatoxin ; US Patent 5981583 where the inhibition of activation of NF-.Kappa.B by caffeic acid phenylester(CAPE) and two analogues of CAPE. Tumor necrosis factor (TNF) activation of NF-.Kappa.B is completely blocked by CAPE in a dose- and time-dependent manner and NF-.Kappa.B activation is inhibited by capsaicin and resiniferatoxin induced by different agents. Reference may be made to Kaleda, William, W, Saleeb, Fouad Z, Zeller, Bary ,L 1988. Coffee decaffeination with caffeic acid; US Patent 4767634 wherein a caffeine containing coffee extract solution is contacted with caffeic acid in the presence of water. The caffeine and the caffeic acid form an insoluble caffeine/ caffeic acid complex and this complex is separated from the coffee extract solution. Reference may be made to copending Indian patent no. 435/del/01 wherein the process deals with rice bran lipase using phenyl boronic acid and the drawback is process works only at higher concentrations.. The main object of the present invention is to provide a process for the preparation of inactivated plant lipases . Another object of the present invention is to provide higher stability of rice bran and wheat germ. Accordingly the present invention provides a process for the preparation of inactivated plant lipases which comprises a. Incubating a mixture of purified active lipase enzyme and a ligand a biomolecule selected from chlorogenic acid , caffeic acid in a molar ratio ranging 1: 1 0 to 1: 200 of protein to ligand at a temperature ranging between 25-40°C for a period of ranging between 10-20 minutes, b. adding a subs' ate selected from triacetin or tributyrin.to the above mixture followed by addition of an activator selected from 10 µl of 0.1 M CaCI2, c. Incubating the above mixture obtained in step (b) at a temperature in the range of 25-40° for a period ranging 3-4 hrs, adding alcohol to the said mixture to obtain the resulting inactivated plant lipase enzyme. In an embodiment of the present invention, the salting out agent is selected from ammonium sulfate and In another embodiment of the present invention, the purification of the lipase enzyme in step (a) of the process may be carried out by dialysis and size exclusion chromatography. In an another embodiment of the present invention, the substrate may be selected from triacetin and tributyrin. In an another embodiment of the present invention the ligand used may be biomolecules selected from chlorogenic acid and caffeic acid. In an another embodiment of the invention, the mixture of active lipase enzyme and the ligand may be added to the substrate at a concentration of at least 5%. In yet an another embodiment of the present invention, the lipase enzyme may be mixed with the ligand in a ratio 1:10, 1:25, 1:50 1:100 , 1:150 and 1:200 mole to mole ratio of protein to ligand. The present invention provides a process for the preparation of inactivated plant lipases using selected biomolecules, which comprises ; a) extracting lipase enzyme from plant source and purifying the said enzyme using a salting out agent to obtain active lipase enzyme ; b) preparing a ligands in the ratio of 1:10, 1:25, 1:50 , 1:100 , 1:150 and 1:200 mole to mole ratio of protein to ligand, c) mixing the said active lipase enzyme and the ligand and adding to it the substrate followed by the addition of activator such as CaCI2 in a concentration of 0.1 M, incubating the mixture thus obtained may be for 3-4 hours to check the activity The different unit operations and conditions involved in the preparation of lipase solution in presence of ligands are given below as flow chart: FLOW CHART Scheme I: (Flow Chart Removed) To approximately 2 mg of wheat germ lipase in 0.02 M sodium phosphate buffer, pH 7.0 is added different concentrations of chorogenic acid ranging from 1:10, 1:25, 1:50, 1:100 , 1:150 and 1:200 mole to mole ratio of protein to ligand incubated for 15 minutes at 37°C in Queue orbital incubator shaker at 50 rpm. After the incubation, to the 2ml of the lipase solution containing different concentrations of the chlorogenic acid, 4ml of the 5% substrate(triacetin or tributyrin ) solution is added in addition to 10 µl of 0.1 M CaCI2 and the reaction mixture is again incubated for 3 hours in queue orbital shaker at 150 rpm. The reaction mixture was checked for remaining enzyme activity by pH-stat method using Mettler Toledo DL12 titrator titrating against 0.05M NaOH . Respective blank solutions where the enzyme was inactivated by the addition of distilled alcohol was also used. The remaining activity was expressed as microequivalents of alkali consumed per mg of protein per hour. FLOW CHART Scheme II: (Flow Chart Removed) To approximately 2 mg of wheat germ lipase in 0.02 M sodium phosphate buffer, pH 7.0 is added different concentrations of caffeic acid ranging from 1:10, 1:25, 1:50, 1:100 ,'1:150 and 1:200 mole to mole ratio of protein to ligand incubated for 15 minutes at 37°C in Queue orbital incubator shaker at 50 rpm. After the incubation, to the 2ml of the lipase solution containing different concentrations of the caffeic acid, 4ml of the 5% substrate(Triacetin or Tributyrin ) solution is added in addition to 10 µl of 0.1 M CaCl2 and the reaction mixture is again incubated for 3 hours in queue orbital shaker at 150 rpm. The reaction mixture was checked for remaining enzyme activity by pH-stat method using Mettler Toledo DL12 titrator titrating against 0.05M NaOH . Respective blank solutions where the enzyme was inactivated by the addition of distilled alcohol was also used. The remaining activity was expressed as microequivalents of alkali consumed per mg of protein per hour. FLOW CHART Scheme III: (Flow Chart Removed) To approximately 2 mg of rice bran lipase in 0.05M sodium phosphate buffer, pH 7.4 is added different concentrations of chlorogenic acid ranging from 1:10, 1:25, 1:50, 1:100 , 1:150 and 1:200 mole to mole ratio of protein to ligand incubated for 15 minutes at 30°C in Queue orbital incubator shaker at 50 rpm. After the incubation, to the 2ml of the lipase solution containing different concentrations of the chlorogenic acid, 4ml of the 5% substrate (Triacetin or Tributyrin ) solution is added in addition to 10 µl of 0.1 M CaCI2 and the reaction mixture is again incubated for four hours in queue orbital shaker at 150 rpm. The reaction mixture was checked for remaining enzyme activity by pH-stat method using Mettler Toledo DL12 titrator titrating against 0.05M NaOH . Respective blank solutions where the enzyme was inactivated by the addition of distilled alcohol was also used. The remaining activity was expressed as microequivalents of alkali consumed per mg of protein per hour. FLOW CHART Scheme IV: (Flow Chart Removed) Activity was calculated from the alkali consumed per mg protein per hour To approximately 2 mg of rice bran lipase in 0.05 M sodium phosphate buffer, pH 7.4 is added different concentrations of caffeic acid ranging from 1:10, 1:25, 1:50,1:100 , 1:150 and 1:200 mole to mole ratio of protein to ligand incubated for 15 minutes at 30°C in Queue orbital incubator shaker at 50 rpm. After the incubation, to the 2ml of the lipase solution containing different concentrations of the caffeic acid, 4ml of the 5% substrate (triacetin or tributyrin ) solution is added in addition to 10µl of 0.1 M CaCI2 and the reaction mixture is again incubated for four hours in queue orbital shaker at 150 rpm. The reaction mixture was checked for remaining enzyme activity by pH-stat method using Mettler Toledo DL12 titrator titrating against 0.05M NaOH . Respective blank solutions where the enzyme was inactivated by the addition of distilled alcohol was also used. The remaining activity was expressed as microequivalents of alkali consumed per mg of protein per hour. The novelty and the uniqueness of the process of the invention is the inactivation of both wheat germ lipase and rice bran lipase using selected biomolecules such as chlorogenic acid and caffeic acid. 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. EXAMPLE -1 (Example Removed) In order to understand the effect of biomolecules on the activity of plant lipases such as wheat germ lipase and rice bran lipase , experiments were carried out at various concentrations of biomolecules. The activity of wheat germ lipase in the presence of chlorogenic acid was checked at 1;50 mole to mole ratio of protein to ligand using 4ml of the 5% substrate and 10 µl of 0.1 M CaCI2. The analysis of the data showed that the enzyme was found to loss 10% of initial activity in presence of 1:50 mole to mole ratio of protein to ligand. EXAMPLE - 2 (Example Removed) The activity of wheat germ lipase in the presence of caffeic acid was checked at 1:50 mole to mole ratio of protein to ligand using 4ml of the 5% substrate and 10 µl of 0.1 M CaCI2. The analysis of the data showed that the enzyme was found to loss 20% of initial activity in presence of 1:50 mole to mole ratio of protein to ligand. EXAMPLE – 3 (Example Removed) The activity of rice bran lipase in the presence of chlorogenic acid was checked at 1:50 mole to mole ratio of protein to ligand using 4ml of the 5% substrate and 10 µl of 0.1MCaCI2. The analysis of the data showed that the enzyme was found to loss 15% of initial activity in presence of 1:50 mole to mole ratio of protein to ligand. EXAMPLE - 4(Example Removed) The activity of rice bran lipase in the presence of caffeic acid was checked at 1:50 mole to mole ratio of protein to ligand using 4ml of the 5% substrate and 10 µl of 0.1 M CaCI2. The analysis of the data showed that the enzyme was found to loss 30% of initial activity in presence of 1:50 mole to mole ratio of protein to ligand. The main advantages of the present invention are: 1) The inhibition of activity of plant lipases by biomolecules leads to the stabilization. 2) This process of inactivation required very low concentrations of inhibitors. 3) Inhibition by biomolecules maintains the other properties of the lipase. 4) The availability of inhibitors We claim: 1. A process for the preparation of inactivated plant lipases which comprises; a) Incubating a mixture of purified active lipase enzyme and a ligand a biomolecule selected from chlorogenic acid , caffeic acid in a molar ratio ranging 1: 1 0 to 1: 200 of protein to ligand at a temperature ranging between 25-40°C for a period of ranging between 10-20 minutes, b) adding a substrate selected from triacetin or tributyrin.to the above mixture followed by addition of an activator selected from 10 µl of 0.1 M CaCl2, c) Incubating the above mixture obtained in step (b) at a temperature in the range of 25-40° for a period ranging 3-4 hrs, adding alcohol to the said mixture to obtain the resulting inactivated plant lipase enzyme. 2. A process as claimed in claim 1 wherein the active lipase enzyme is from plant source selected from rice bran lipase, wheat germ lipase. 3. A process as claimed in claims (1) to (3), wherein the mixture of active lipase enzyme and the ligand to the substrate is at a concentration of at least 5%. 4. A process as claimed in claims (1) to (4), wherein the lipase enzyme is mixed with the ligand in the mole to mole ratio of group selected from 1:10, 1:25, 1:50, 1:100 , 1: 150 and 1 :200 of protein to ligand. 5. A process as claimed in claims (1 ) to 5 wherein the incubation in step (a ) and (b) is carried out at a temperature in the range of 30-37°C, 6. A process as claimed in claims 1- 6 wherein the molar ratio of protein to ligand is 1:50. 7. A process for the preparation of inactivated plant lipases substantially as herein described with reference to the examples accompanying this specifications..

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