Soybean lecithin : acetone insoluble residue fractionation and their volatile components

El residuo insoluble en acetona fue aislado de la lecitina de soja. Este residuo fue fraccionado por solventes en cuatro fracciones: soluble en ácido acético, insoluble en ácido acético, fase benceno y fase insoluble en benceno. Concerniente a la constitución de los fosfolípidos de estas cuatro fracciones, se encontró que la primera fracción contiene PC, PE y PI en porcentajes del 56.0, 21.6 y 19.0 respectivamente. La segunda fracción tuvo 39% PC y 60% CER, junto a algunas trazas de PE y PI. La fase benceno está constituida principalmente por PC con algunas trazas de PE. La última fracción tuvo 80.6% CER y 20% PC. La composición en ácidos grasos de estas cuatro fracciones junto a los solubles en acetona, aceite de soja crudo y desgomado y fosfolípidos totales fueron registradas. Por lo general, se encontró que los ácidos grasos mayoritarios saturados e insaturados fueron el palmítico y linoléico. Los componentes volátiles de estas muestras excepto la insoluble en ácido acético fueron determinadas. Cuarenta y nueve compuestos fueron separados. Treinta y dos componentes incluyendo aldehidos alifáticos, cetonas, alcoholes, ésteres y ácidos fueron identificados. Los aldehídos y cetonas mostraron un cambio en las siete muestras. Estos aumentaron por el desgomado. El 4,5-dimetilelisoxazol tuvo un fuerte flavor a lecitina, así puede usarse como indicador para el proceso de desgomado. El 2-pentilfurano mostró una disminución significativa por el desgomado. Otros compuestos, tales como ésteres y alcoholes no tuvieron efectos distintivos sobre los productos volátiles a lo largo del proceso.


INTRODUCTION
The soybean is a commercial source of lecithin (1).Worldwide consumption of this by-product of soybean processing is estimated at 10,000 tons / year (2).Industrially, lecithin is removed by treating the crude oil with water (2), inorganic and organic acids (3) or acetic anhydride (4) at elevated temperatures; the gums precipitate from the oil and are removed by centrifugation or sedimentation.
The term "lecithin" as used to day refers to the material obtained by degumming crude vegetable oils and drying the hydrated gums.Lecithin, then, consists not only of a mixture of phospholipid components removed from the degumming process.

Preparation of soy lecithin
Soybean oil (2.000 g) was charged into a 3 liter round bottom flask with a stirring shaft and a paddle shaped Teflon impeller 7.5 cm long driven by a variable speed motor.The oil was purged with nitrogen through a sinter glass stick for minute and brought to the desired temperature under a nitrogen Grasas y Aceites blanket; then the motor was started, and the desired amount of distilled water was added.When degumming was completed, the mixture was cooled to 40 o C and the soy lecithin was separated by centrifugation at 1.900 rpm for 15 minute.The degummed oil was removed by decantation.Soy lecithin was purified by extraction of the rest of soybean oil with n-hexane and was separated by filtration and washed several times with the same solvent and dried under vacuum at 40 o C while bubbling nitrogen then kept under nitrogen in refrigerator.

Isolation of the acetone -insoluble residue
Fifty grams of soy lecithin were dissolved in the least amount of ether and the solution then filtered through Whatman No. 1 filter paper on a Buchner funnel.Twenty volumes of cold acetone were added to the filtrate, small portion at a time while contineous stirring.The solution was then allowed to settle over night in the refrigerator.The acetone -insoluble residue was centrifuged and then successively washed with voluminous amounts of cold acetone.The acetone insoluble residue was in a small volume of diethyl ether and reprecipitated with acetone in the same manner.

Solvent fraction of the acetone -insoluble residue
The acetone insoluble residue was slurried with glacial acetic acid (1: 3, v/v) in a warring blender.The suspension was poured into 350 ml glacial acetic acid in a 2 liter beaker while contineous stirring.The suspension was allowed to stand at room temperature for 2 hour and then filtered through Whatman No. 1 filter paper on a Buchner funnel.The residue was re-extracted with 200 ml of glacial acetic acid in the same manner.The acetic acidsoluble (fraction I) was evaporated under vacuum at 45 o C while bubbling nitrogen in the solution and the residue was dissolved in 20 ml of hot distilled water and re-evaporated in the same manner, and the process repeated to get ride of all acetic acid vapours.The acetic acid -insoluble (fraction II) was suspended in 100ml of benzene and 50 ml distilled water in a centrifuge bottle.The bottle was shaken vigorously for 15 minutes and then centrifuged.The benzene phase was re-extracted twice with 75 ml portions of benzene.The combined benzene extracts, after being dried over anhydrous Na 2 SO 4 (fraction III) and the benzene -insoluble phase, aqueous phase, (fraction IV) were evaporated as described previously in acetic acidsoluble fraction I.

Fatty acid composition of Crude soybean oil, Degummed oil, Acetone soluble, Acetic acid soluble and total phospholipid, besides Benzene insoluble phase and Benzene phase
The component fatty acids of these seven samples were converted to their methyl esters by esterification ( 5), the mixed methyl esters were subjected to gas liquid chromatographic (GLC) analysis (6).

Volatile components of Crude soybean oil, Degummed soybean oil, Acetone soluble and Acetic acid soluble besides Total phospholipid, Benzene insoluble phase and Benzene phase
The volatile components of soluble in acetone, soluble in acetic acid, benzene phase and benzene insoluble phase fractions were prepared.Volatile components of crude soybean oil, Degumming soybean oil and total phospholipid were also collected for comparison.
The identification of the components of each flavour was done using coupling gas liquid chromatographymass spectrometry (Varian 1400-Mat 112).Temperature of ion source of mass spectrometry is 200 o C under reduced pressure 10 -6 torr and electron volt 70 ev.

Gas liquid chromatography
It was done using Varian 3700 dual flame ionization detector under the following conditions: Column package 20% Diethylene glycol succinate (DEGS) on chromosorb W( 60-80 mesh), column length 6 feet with internal diameter 1

Preparation of the Aroma concentrate
The aroma concentrate of each sample was as follow : Suitable amount of the sample was heated on silicon oil bath at 190 o C in 500 ml round bottom flask under reduced pressure (about 30 mm Hg).The aroma was collected in traps cooled to -20 o C using crushed ice-salt-acetone mixture.The collected distillate was thrice extracted with peroxide free ether, and the combined extracts were dried over 320 Grasas y Aceites anhydrous sodium sulphate.The extract was filtered and ether was then removed by distillation at 40 o C to obtain the aroma concentrate.It can be seen from table I that: 1.The acetic acid soluble contain PC, PE and PI in percentages of 56.0, 21.6 and 19.0 respectively.
2. The benzene phase mainly all PC with some traces of PE.
3. The major part of the benzene insoluble phase was CER which makes 80.6 % and PC makes 20 %.Table II shows the fatty acid composition of these compounds.

Fatty Acid
The major fatty acids are linoleic acid and palmitic acid.The ratio of saturated-unsaturated fatty acids ranges from 1:1.5 to 1:5.85.
Fourty nine compounds were separated.Thirty two compounds including aliphatic aldehydes, ketones, alcohols, esters and acids were identified.
Among these compounds, aldehydes are formed to be present in high concentration reaching more than 55 % in crude oil 2,4 undecadienal and 2,4,6 dodecatrienal has the highest concentration 11.94 % and 15.64 % respectively among them.
The short chain aliphatic aldehydes showed a remarkably decrease for the total phospholipid.
The lack of these aldehydes (C 3 -C 12 ) in the volatile decomposition products of phospholipid may result from the immediate reaction of the aldehydes produced from the fatty acid moieties towards the amino group of phospholipids, especially phosphatidylethanolamine (7,8).2, Pentylfuran and 4,5 Dimethyllisoxazole were identified in the volatile components of total phospholipid.2,4-Dimethyllisoxazole may derive from the reaction of 1,3-dicarbonyl compound with the decomposition products phosphatidylethanolamine (9).
The mechanism that forms ketones, aldehydes, alcohols and esters through the decomposition of hydroperoxides of the unsaturated fatty esters has been reviewed (10).The aliphatic aldehydes (saturated and unsaturated) showed a remarkable increase and in particularly 2,4 undecadienal (21.25 %) and 2,4,6 dodecatrienal (23.01 %) among the volatile compounds of degummed soybean oil.This was in agreement with Kim et al. (11), who found that oil free  soybean lecithin enhance the formation of aliphatic aldehydes.
The volatile components of acetone soluble have high concentration of aldehydes, in particularly 2,4,6 dodecatrienal (41.29 %).This fraction is neutral lipid, in which by heating gives rise to aliphatic aldehydes, ketones, esters, and alcohols.The large number of aldehydes with these high concentrations have been reported in the volatile decomposition products of neutral lipids (12,13).Ketones are forming the secondary remarkable concentrations after aldehydes, in the volatile components of acetone