Effect of interesterification on glyceride structure

Se ha llevado a cabo el proceso de interesterificación en aceite de soja ácido oleico (2:1 w/w) a diferentes concentraciones de níquel como catalizador (0.4 y 0.6%), a una temperatura de 90°C y a un tiempo de batido de 4 horas. La hidrólisis de la estructura glicerídica individual se hizo usando lipasa pancreática para obtener 2-monoglicéridos. El cálculo se llevó a catx) para determinar la estructura glicerídica del aceite de soja interesterificado.


INTRODUCTION
Interesterification is a process that is used to modify the structure and composition of oils and fats to improve their physical and nutritional properties (1,2).Conventionally, the interesterification reaction is promoted by sodium metal or sodium alkoxide to catalyze acyl migration between triglyceride molecules and to produce randomly distributed fatty acyl residues among the triglyceride molecules (3).
In recent year, the use of lipases as biocatalysts for interesterification reactions has become of great industrial interest for the production of useful triglyceride mixtures.This is mainly because of the specificity of these enzymes with respect to glyceride positions and fatty acid types (4).Lipases are known to catalyze mild esterification reaction with the formation of specific compounds that are easy to isolate without molecular distillation (5).
Akoh and co-workers (6) described synthesis of monoglycerides in organic solvent by lipase.This enzyme successfully catalyzed the esterification of glycerol with oleic acid or eicosapentanoic acid (EPA) in hexane.The pancreatic lipase, which possesses near absolute specificity for hydrolysis of the sn 1,3 position in triglycerides, shows a difference in the distribution pattern of fatty acids in the resulting 2-monoglycerides of interesterified and hydrogenated fats.The pancreatic lipolysis method, followed by examination of the fatty acids of the 2-monoglycerides, is a method for detecting an interesterified fat (7).
Lipase can be used in several ways in the modification of triglycerides.The physical properties of naturally occurring triglycerides depend not only on saturation or unsaturation of the attached acyl groups, but also on the position of these groups.In chemical interesterification, it is difficult to introduce the desired acyl group into the desired position on glycerol because the reaction occurs randomly (8).
Mottson and Lutton (9) studied the specific distribution of the fatty acids in the glyceride of animal and vegetable fats by means of pancreatic lipase, which removed a-substituted fatty acids predominently.
One application of the lipase catalyzed interesterification of triglycerides which has been studied is the preparation of cocoa butter equivalents.Normally the starting material is palm oil mid fraction and the purpose of the lipase catalyzed reaction is to introduce more stéarate into the triglycerides of the palm oil mid fraction.The main component of palm oil mid fraction is the triglyceride 1,3 dipalmitoyl-2-oleoyl-glycerol (POP), which can be converted to 1-palmitoyl-2-oleoyl-3-stearoyl-glycerol and 1,3-distearoyl-2-oleoyl-glycerol by the interesterification reaction.The result is a fat with a triglyceride composition resembling cocoa butter which can be used as cocoa butter equivalent in the chocolate and confectionary industry As it is the fatty acids in position 1 and 3 which should be replaced, the 1,3 specific lipases are the enzymes of choice the reaction medium normally consist of a mixture of the triglyceride to be converted and the stéarate component either as free acid or an ester, in a nonpolar organic solvent (10).
In this article of work the glyceride structure of interesterified soybean oil under different conditions of temperature, time of stirring and nickel catalyst percentages will be investigated in an attempt to give a clear picture on its fatty acids distribution in comparison with soybean oil.

MATERIALS AND METHODS
-Bleached soybean oil and oleic acid were obtained from Cairo Company for Oils and Soaps.
-Chemical interesterification was carried out with different concentration of nickel catalyst (0.4 and 0.6%) at 90°C and 4 hours of stirring.Soybean oil and oleic acid were in the ratio 2:1 w/w.
-Source of lipase: pancreatic lipase was obtained from Sigma Chemical Company.
Procedure for lipase hydrolysis: Hydrolysis of triglyceride fractions was carried out to obtain the 2-monoglyceride (11).
Approximately 1.0 gm of interesterified soybean oil was transferred to a tipped bottom flask, and the temperature was adjusted to 40° ± 0.5°C.To the sample, 10 ml of 1.2 M ammonium chloride-ammonia hydroxide (NH4CI -NH4OH ) buffer, adjusted to pH, 8.5, 2 ml (22 %, w/v) solution of calcium chloride hexahydrate and 0.1 ml of a 25 % w/v solution of bile salt sodium taurocholate were added.Pancreatic lipase (0.3 gram) was added with stirring and the pH was maintained at 8.5 during hydrolysis.After 30 minutes about 60 or 70 % of original natural triglyceride was converted into 2-monoglyceride.Then the reaction was stopped by the addition of 4N hydrochloric acid to bring the pH to 1.0.The reaction mixture was extracted with diethyl ether and the ethereal solution was dried over anhydrous sodium sulphate, filtered and evaporated under reduced pressure.The 2-monoglycerides were separated from the other lipids and free fatty acid by preparative thin layer chromatography (12) on silica gel G with hexane and diethyl ether (60:40).
The 2-monoglycerides were extracted with chloroform and then convert to methyl esters according to Metcalfo and Schmitz (13) and were examined by gas liquid chromatography under these conditions: -Adual flame ionization detector Varian 3700, with packed column 20 % diethylene glycol succinate (DEGS) on chromosorb W 60-80 mesh.The column length 6 feet with internal diameter 1/8 inch, carrier gas flow rate (He) 30 ml/min., hydrogen flow rate 30 ml/min and air flow rate 300 ml/min.

RESULTS AND DISCUSSION
Concerning the study the fatty acid constituents of soybean oil before interesterification (control) and after carrying the reaction at 90°C for four hours of stirring using 0.4 and 0.6 % nickel catalyst as well as those of the 2-monoglycerides obtained by lipase hydrolysis are recorded in table I.We trace that linoleic and linolenic acids of interesterified soybean oil samples and their 2-monoglycerides are decreased comparing to the control samples.Whereas oleic and stearic acids are increased in interesterified oil while oleic is decreased and stearic is increased in 2-monoglycerides of the interesterified samples.This means that the fatty acid composition of the 2-monoglycerides are different from those of the corresponding whole triglycerides which agree with the view of Roy and Bhattacharyya (7).The results recorded in tables ll-V showed the 2-monoglycerides regarding the type of glycerides.We notice that 2-olein glycerides with stearin at 1and/or 3-positions are more than that of 2-stearin glycerides with olein at 1-and/or 3-positions, while 2-linolein with linolenolein at 1-and/or 3-positions and vice versa are small quantities.This mean that the 2-monoglycerides produced by pancreatic lipase hydrolysis of soybean oil triglycerides contain mostly C18 unsaturated fatty acids (11) and the remaining fatty acids are randomly distributed among sn 1,3-positions of the triglycerides and these findings are in accord with the positional distribution theory suggested by Van Der Wal (15), Coleman and Fulton (14), Cunstone (16), Youngs (17), Roy and Bhattacharayya (7).Table VI, showed the triglyceride percentages in terms of the four main glyceride categories, namely trisaturated (GS3), disaturated (GS2U), monosaturated (GSU2) for the interesterified soybean oil samples.It is clear that the GS3 values is 11.707 and 13.209 % Vol.49.Fase.5-6(1998) 417 using 0.4 and 0.6 % nickel catalyst, respectively, which is lower than the expected one when correlated to the content of total saturated fatty acids (Ci6:o and Ci8:o) of interesterified soybean oil sannples about 54% table I.This fact agrees with the findings of Kartha (18)(19)(20)(21) concerning restricted random distribution theory.The formation of trisaturated glycerides is believed to be due to the presence of saturated fatty acids in the 2-position (10) 1,2-diglycerides are chemically unstable, and therefore they undergo either spontaneously or enzymatically added acyl migration to form 1,3 diglycerides (22,23).Generally, an interesterification of soybean oil using 0.4% nickel catalyst give the proper stability to the oil.So from the economical point of view it is more practical in application.