Lecithins enzymatic hydrolysis

The phosphoric acid esters of diacyl glycerides, phospholipids, are important constituents of cellular membranes. Lecithins (phosphatidyl cholines) from egg white or soybeans are often added to foods as emulsifying agents or to modify flow characteristics and viscosity. Phospholipids have very low vapor pressures and decompose at elevated temperatures. The strategy for analysis involves preliminary isolation of the class, for example by TLC, followed by enzymatic hydrolysis, derivatization of the hydrolysis products, and then GC of the volatile derivatives. A number of phospholipases are known which are highly specific for particular positions on phospholipids. Phospholipase A2, usually isolated from snake venom, selectively hydrolyzes the 2-acyl ester linkage. The positions of attack for phospholipases A, C, and D are summarized on Figure 9.7 (24). Appropriate use of phospholipases followed by GC can thus be used to determine the composition of phospholipids. 

In some cases separation of lecithins from phosphatidyl ethan-olamines and phosphatidyl serines is carried out prior to enzymatic hydrolysis. Composition of the phospholipids is virtually always determined finally by GLC of their fatty acid methyl esters. 

The simplest method for modifying natural (crude) lecithin is the addition of a non-reactive substance. Plastic lecithins are converted to fluid forms by adding 2% to 5% fatty acids and/or carriers such as soybean oil. If the additives react with the lecithin to alter the chemical structure of one or more of the phospholipid components, the resulting product is referred to as a chemically modified lecithin. Modification can also be achieved by subjecting lecithin to partial controlled enzymatic hydrolysis. Finally, refined lecithin products can be obtained by fractionating the various phospholipid components. 

Modified lecithins. Lecithins may be modified chemically, e.g., hydrogenation, hydroxylation, acetylation, and by enzymatic hydrolysis, to produce products with improved heat resistance, emulsifying properties, and increased dispersibility in aqueous systems (7, 58, 59). One of the more important products is hydroxylated lecithin, which is easily and quickly dispersed in water and, in many instances, has fat-emulsifying properties superior to the natural product. Hydroxylated lecithin is approved for food applications under Title 21 of the Code of Federal Regulations 172.814 (1998) (60). 

Cmde lecithin contains a number of functional groups that can be successfully hydrolyzed, hydrogenated, hydroxylated, ethoxylated, halogenated, sulfonated, acylated, succinylated, ozonized, and phosphorylated, to name just a few possibilities (1). The only chemically modified food-grade products produced in significant commercial quantities at the present time are the ones obtained by hydroxylation, acetylation, and enzymatic hydrolysis (58). Hydroxylated or acylated lecithins represent chemical modifications to improve the functionality in water-based systems. 

Haas et al. (162) have studied enzymatic phosphatidylcholine hydrolysis in organic solvents by examining selected commercially available lipases. Enzymatic hydrolysis of oat and soy lecithins, and its effect on the functional properties of lecithin, was investigated by Aura et al. (163). The phospholipase used was most effective at low enzyme and substrate concentrations. 

N. E. Gabriel, N.V. Agman, and M. F. Roberts. Enzymatic-hydrolysis of short-chain lecithin long-chain phospholipid unilamellar vesicles sensitivity of phospholipases to matrix phase state. Biochemistry, 1987, 26, 7409-7418. 

CAS 85711-58-6 EINECS/ELINCS 288-318-8 Synonyms Hydrolyzed lecithin Lecithins, hydrolyzed Definition Prod, obtained from the enzymatic hydrolysis of lecithin Properties Amphoteric... 

The enzymatic hydrolysis of lecithin to fatty acids, glycerol, phosphoric acid, and choline requires the participation of some four enzymes. The enzymes attacking the various bonds of a lecithin molecule are summarized in Table XIV. The bonds which are cleaved are referred to by number in the lecithin formula shown below the same numbers are used in Table XIV to indicate the respective enzymes involved. The cleavage of the fatty acid-glycerol bond, as represented by diagonal lines 1 and 2, is based on hydrolysis studies of simple esters. Diagonal lines 3 and 4 are only provisional, as presumably either a P—0 or C—O bond to phosphate could be cleaved. 

In principle chemical hydrolysis of the fatty acids from the phospholipid molecules is possible. However, this reaction is not applied on a plant seale, because enzymatic hydrolysis is the preferred state-of-the-art process. Chemical partial hydrolysis can be made with various strong acids, but the modified lecithins usually have a dark brown to black colour, forming an obstacle for successful industrial use in many applications. 

Direct GC-MS. Phosphatides isolated from lecithin may be analyzed after isolation and enzymatic hydrolysis of the phosphorus-containing substituent. The diglycerides are analyzed by GC-MS of the TMS derivatives (124). 

Finally, the enzymatic nature of CPIA-cholesterol ester formation will be briefly mentioned. None of the enzyme preparations of three known biosynthetic pathways for cholesterol esters, namely, acyl-CoA cholesterol Q-acyltransferase (ACAT), lecithin cholesterol 0-acyltransferase (LCAT), nor cholesterol esterase, was effective in producing CPIA-cholesterol ester from the Ba isomer or CPIA. In contrast, the 9,000 g supernatant or microsomal fractions from liver or kidney homogenate were found to be capable of producing CPIA-cholesterol ester without the addition of any cofactors. As substrate, only the Ba isomer was effective, and none of the 3 other fenvalerate isomers nor free CPIA was effective. The hepatic enzyme preparation also catalyzed hydrolysis of fenvalerate, and in this case all the 4 isomers were utilized as substrates. These facts imply that CPIA-cholesterol ester is formed from the Ba isomer through a transesterification reaction via intermediary acyl-enzyme complex. 

HPLC is the most common technique applied to the determination of the chemical composition of lecithin. Normal phase HPLC is convenient for the determination of the major constituents (i.e., phosphatidylcholine, phosphatidylethanolamine, etc), as described in Chapter 7. P NMR is also suitable for this analysis, as discussed in Chapter 14. The biochemical literature contains many enzymatic methods, mainly for specific determination of phosphatidylcholine and its hydrolysis product, choline (32). For instance, phosphatidylcholine can be hydrolyzed by phospholipase C to a diacylglycerol and the phosphate ester of choline, which itself can be hydrolyzed by alkaline phosphatase to form choline and phosphate ion. Alternatively, action of phospholipase D on phosphatidylcholine yields phosphatidic acid and choline. These methods are not applied to analysis of the commercial lecithin used as a surfactant. 

In addition, it was desired to explore the effects that these small changes in intermolecular distance had on the enzymatic susceptibility of these lecithins to hydrolytic enzymes such as phospholipase A [3-5], a potent hydrolytic enzyme found in cobra venom. Thus, microgram quantities of enzyme were injected under this monolayer. By measming the rate of change of surface potential, one can indirectly measine the rate of reaction in the monolayer. It is assumed that these quantities [i.e., change in surface potential A(AV) and the extent of reaction] are proportional to each other. The kinetics of hydrolysis, as measured by a decrease in sinface potential, were studied for each lecithin monolayer as a fimction of initial surface pressure and are shown in Fig. 3 [6]. It was foimd that initially the reaction rate... 

---