Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing organic compound containing a metal or atom other...
Reexamination Certificate
2001-03-28
2002-06-11
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing organic compound containing a metal or atom other...
C435S128000, C435S197000, C435S198000
Reexamination Certificate
active
06403344
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an enzymatic process for the preparation of an acetylated phospholipid. The invention particularly relates to an enzymatic process for the preparation of an acetylated phospholipid from vegetable lecithins such as soybean lecithin, rapeseed lecithin etc., and animal lecithins like egg yolk lecithin or pure phosphatidylethanolamine isolated from the above lecithins. The commercial lecithins or phosphatidylethanolamine are acetylated by using vinyl acetate as acylating agent in presence of lipase from
Mucor miehei
having 1,3-position specificity as catalyst.
BACKGROUND OF THE INVENTION
Commercial lecithin is an important co-product of oil processing obtained during degumming step. For example, soybean lecithin is a complex mixture and comprises of phospholipids and triglycerides, with minor amounts of other constituents like phytoglycolipids, phytosterols, tocopherols and fatty acids. The major phospholipids present in vegetable lecithins are phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol. The egg yolk lecithin contains phosphatidylcholine and phosphatidylethanolamine as major phospholipids. Lecithin has potential as a multifunctional additive for food, pharmaceutical and industrial applications. The primary usage of lecithin in food is as an emulsifier. (Dashiell, G. L., in Lecithins: Sources, Manufacture and Uses, edited by B. F. Szuhaj, American Oil Chemical Society, Champaign Ill., 1989, p. 213). Acetylated lecithin exhibits improved fluid properties, improved water dispersability, and is effective oil-in-water emulsifiers for a wide variety of food formulations. (J. S. Schmidt and F. T. Orthoefer in Lecithins, B. F. Szuhaj and G. R. List eds., American Oil Chemists'Society, Champaign, Ill., 1985. p.183-212). Acetylation occurs primarily on the amino group of phosphatidylethanolamine. Moderately and highly acetylated lecithins are resistant to heat and can be repeatedly heated and cooled without darkening. The intended uses of minimally acetylated products are in infant foods coffee whiteners, meat sauces and gravies, and for oil-in-water cosmetic emulsions. Moderately and maximally acetylated products are used in cheese sauces, shortenings, and as release agents in pumpable and aerosol formulations, (Bailey's Industrial Oil and Fat Products, edited by Y. H. Hvi, Vol 1, 5
th
Ed., John Wiley & Sons, N.Y., 1966, p.341).
There have been sporadic attempts to prepare acetylated lecithin from soybean lecithin. Unilever [Netherlands Patent 6,700,366 (1967)] prepared acetylated lecithin by react soya lecithin (100 g) containing 65% phosphatides with 2 g of acetic anhydride at 70° C. The mixture was stirred for 25 minutes and the acetic acid was distilled at 3 mm yielding 101 g of a mixture containing acetylated lecithin. Central Soya Company Inc., [U.S. Pat. No. 3,301,881 (1967)] in their studies used carboxylic acid anhydride like acetic anhydride for the acetylation of phospholipids from vegetable lecithins. J. Eichberg [U.S. Pat. No. 3,359,201 (1967)] also reported a similar methodology to prepare acetylated lecithin using acetic anhydride as acylating agent. According to R. Aneja and J. S. Chadha [Fett Seifen 73, 643-651 (1971)] acetylation was incomplete with acetic anhydride even after prolonged periods under reflux. However, in the presence of tertiary amine, the reaction was rapid and essentially complete in a few minutes at room temperature. Accordingly, Aneja, R. [U.S. Pat. No. 3,704,254 (1972)] used similar methodology by stirring soybean lecithin (100 g) and acetic anhydride (4 g) together vigorously under nitrogen at 74° C for 2 hr followed by tertiary amine treatment. The water dispersability of soybean lecithin was improved by acetylation with acetic anhydride at 170° F. followed by hydroxylation with 35% hydrogen peroxide [U.S. Pat. No. 3,962,292 (1976)] and the product was dried and deodorized at 185-190° F. and 28 mm Hg vacuum. Crude soybean phosphatide (1 kg) was dried and dissolved in 4 liters of dichloromethane and mixed with 230 ml acetic acid and 200 g freshly calcinated basic aluminium oxide and refluxed for 4 hours. About half of the phosphatidylethanolamine was acetylated. The aluminium oxide was filtered and the solvent and excess acetic acid were removed under vacuum [Ger Pat. 2,615,120 (1977)]. Guenther, B. R. also prepared acetylated lecithin [Eur. Pat. 54,768 (1982) and U.S. Pat. No. 4,443,378 (1984)] by reacting 400 g of soybean phosphatide with 24 g of acetic anhydride at 50° C. for 1 hr. Soya lecithin was acylated with oleoyl chloride and triethylamine in 96% yield. N-Acyl phosphatidylethanolamine is useful as antioxidant for unsaturated fatty acids [Ger Pat. 4,141,842 (1993)]. Dashiell, G. L. and William E., (U.S. Pat. No. 4,479,977 (1984) prepared acetylated lecithin by treating lecithin with 2-5% of acetic anhydride and claims that the lecithin-based release agents with superior resistance to darkening, reduced production of objectionable odors, and retention of chemical integrity are produced (release agent for diverse applications). Liposome dispersions are prepared from acylated phospholipids in which the amino group of cephalin was monoacylated with a dicarboxylic acid. [U.S. Pat. No. 4,983,397 (1991)]. Phosphatidylethanolamine was reacted with dodecaniedioic acid in dry dichloromethane in presence of dicyclohexylcarbodiimide, triethylamine and absolute methanol and the mixture was incubated for 24 hours at 40° C. [U.S. Pat. No. 5,064,817 (1991)] to study the Phospholipase A
2
inhibitor activity.
Thus many of the processes reported used only acetic anhydride as acylating agent. Some processes involve the use of acetic anhydride/carboxylic acid anhydride as the acylating agent and triethylamine as catalyst. In another method acetic acid as acylating agent and calcinated basic aluminium oxide as catalyst were used. The conversion of phosphatidylethanolamine to N-acetyl phosphatidylethanolamine remains incomplete when only acetic anhydride is used for the reaction [R. A. Aneja, J. S. Chadha, J. A. knaggs, Biochim. Biophys. Res. Commun 36, 401 (1969)].]. However, R. Aneja and J. S. Chadha [Fett Seifen Anstrichmittel 73, 643-651 (1971)] reported that the reaction was rapid in the presence of tertiary amine as catalyst. Another disadvantage of chemical acetylation is O-acetylation reaction of free hydroxyl groups of phosphatidylinositol present in lecithin in addition to N-acetylation of phosphatidylethanolamine [R. Aneja and J. S. Chadha, Fett Seifen Anstrichmittel 73, 643-651 (1971)]. In general, chemical methods suffer several drawbacks such as low yields, and decomposition products that result in dark colored reaction products.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide an enzymatic process for the first time for the preparation of acetylated phospholipids from vegetable lecithins such as soybean lecithin, rapeseed lecithin etc., and animal lecithins like egg yolk lecithin or pure phosphatidylethanolamine isolated from the above lecithins which obviates the drawbacks of the processes described above.
Another object of the present invention is to develop a simple method to use vinyl acetate as acylating agent, which can be used as a solvent also.
Yet another object of the present invention is to provide an enzymatic method using lipase from
Mucor miehei
having 1,3-position specificity, which can be carried out under mild conditions compared to existing chemical methodologies and which is also an eco-friendly process.
Yet another object of the present, invention is to acetylate amino group of phosphatidylethanolamine selectively without acetylation of hydroxyl group of phosphatidylinositol present in lecithin. This is possible only in the enzymatic acetylation using 1,3-specific lipase as described in the present invention. 1,3-Specific lipase does not allow the secondary
Marellapudi Sri Lakshmi Karuna
Penumarthy Vijayalakshmi
Rachapudi Badari Narayana Prasad
Vemulapalli Vandana
Council of Scientific and Industrial Research
Ladas & Parry
Lilling Herbert J.
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