3.3.2.2 Phospholipase A2

Phospholipase A2 (EC 3.1.1.4) specifically catalyzes the hydrolysis of acyl at number Sn-2 position of phospholipids to produce lysophospholipids and fatty acids. Modified soybean phospholipids exhibit obviously improved hydrophilic and emulsifying properties. They can maintain good emulsifying property under conditions of high or low temperatures or low pH or various salt concentrations. Lysophospholipids are applied in bakery food. They form complexes with amylose and retard aging of breads effectively. Lysophospholipids are two times the price of ordinary phospholipids but they have the advantages of smaller dosage, better effect, oxidative stability and antibiotic property. They are industrially produced in Japan and America. The process is as follows: phospholipids are subjected to moisture content adjustion previously and then added into the solutions with phospholipase A2 of 0.02% (w/v) and calcium chloride of 0.3% (w/v) with stirring. The temperatures are 50-55 degrees Celsius and the reacting time is 7h-9h. The hydrolyzing degree reaches 35%-40% when the acid value (AV) is in the range of 33-30. The following procedures are required to obtain powdery lysophospholipids: concentration under reduced pressure, pressure filtration, washing with acetone, solvent removal under reduced pressure and vacuum drying (Song et al., 2007).

#### 3.3.2.3 Phospholipase C

Phospholipase C acts on phospholipids to produce diglyceride, phosphoinositide, phosphocholine, phosphoethanolamine and phosphoric acid etc. Diglyceride is a bioactive substance which acts as the second messenger in cell signaling transmission and affects the cell metabolism. There are three kinds of specificities of microbial phospholipase C: the first one specifically hydrolyze PI into diglyceride and cyclic phosphoinositide; the second one specifically hydrolyze sphingomyelin and the third one has relatively wider specificity and takes PC as the optimal substrate (Song et al., 2007).

#### 3.3.2.4 Phospholipase D

Phospholipase D (EC 3.1.4.4) can hydrolyze PC into phosphatidic acid and choline. In microwater system with alcohol, phospholipase D can catalyze transacylation which results

Soybean Phospholipids 495

temperature to 80 degrees Celsius decreased the extraction yield and PC content which was attributed to decrease of solubility and selectivity of solvents to PC as the solvents density

PI causes concern as it is involved in the transmission of messages in the cell. PI plays and important role in maintaining normal physiological functions of central nervous system, especially in regulating calcium homeostasis. PI on cell membrane can be hydrolyzed by phospholipase C into 1,4,5-triphosphate inositol that goes into intracellular aqueous phase as the second messenger and 1,2-diacylglycerol that stays in the cell wall. These two substances synergetically induce cell reactions such as contraction, secretion, metabolism

Soybean phospholipids are rich in PI. PI is a white amorphous solid with its sodium salt a crystal and is wet-sensitive. PI is soluble in water, chloroform and benzene, slightly soluble in methanol, diethyl ether and petroleum ether, insoluble in acetone, ethanol and water. It

The solvent method is conducted to isolate and purify phospholipids and increase the content of a certain constituent with single or mixture of such solvents as methanol, ethanol,

The deoiled soybean phospholipids are extracted with appropriate amount of ethanol. The induced ethanol-insoluble phase is vacuum dried to obtain a mixed phospholipids with more PI and less PC which are dissolved in n-hexane before adding 55% ethanol with sodium acetate. The mixture is put into the separating funnel, shaked, allowed to rest and layered. The same procedure is carried out again except that the 55% ethanol is sodium acetate free. The PI obtained is 40%-50% pure. If sodium acetate is replaced by aqueous ammonia with a 8.0 pH and the ethanol concentration increased to 90%, PI of 85% pure can

Purer PI can be obtained by some chemical reaction methods. Soybean phospholipids containing 40% mixed phospholipids are dissolved in such organic solvents as anhydrous pyridine, acetonitrile, dimethyl formamide (DMF) and dimethylsulphoxide (DMSO) etc. Chloride dimethyl tertiary butyl silicon, chloride trimethyl silicon or allyl bromide are added into the mixture to protect hydroxyls of PI by reacting with them. Then PI is isolated from the mixture with solvents such as acetone or ethanol-acetone and hydrolyzed by alkali or acid at room temperature to remove the blocking groups and recover the hydroxyls of PI. PI obtained this way is 98% pure and applied in treating of central nervous system disorder

The phospholipids mixture is dissolved in the mixture of chloroform and methanol in the 1:1 (v/v) ratio before adding aluminium oxide. The eluate contains PC, lysophospholipids, neutral lipids and glycolipids etc. Residues are washed and extracted with the mixture of chloroform, methanol and 1% hydroxyl ammonium acetate in the 1:1:0.3 (v/v/v) ratio and the eluate is loaded on silica column of which the dimension is 30cm. Neutral lipids are eluted with chloroform; glycolipids and PE are sequentially eluted with chloroform and

can be easily oxidized upon exposure to the air (Deng et al., 2003).

isopropyl ketone, acetone, n-hexane and chloroform etc.

decrease at high temperatures.

and proliferation etc.

**4.2.1 Solvent method** 

be obtained with the same method.

**4.2.2 Column chromatography** 

(Deng et al., 2003).

**4.2 Soybean phosphatidylinositol (SPI)** 

in exchange of primary or secondary hydroxyl of some molecules with ethanolamine or choline groups of phospholipids and formation of new phospholipids. This character is called phospholipids' transfer characteristic or base exchange reaction of phospholipase D (Song et al., 2007).
