Effect Of Heat Treatment On Soybean Phospholipase D Activity Determined By Two Methods

Effect of Heat Treatment on Soybean Phospholipase D Activity Determined by Two Methods


Tosi, E. (*), Ballerini, G., Ré, E., and Tapiz, L.


Centro de Investigación y Desarrollo en Tecnología de los Alimentos
Universidad Tecnológica Nacional - Facultad Regional Rosario
E. Zeballos 1341, S2000BQA, Rosario, Santa Fe, Argentina






(*) Author to whom correspondence must to beaddressed
Email address: etosi@ciudad.com.ar



Abstract
Non-hydratable phosphatides are formed by the phospholipase D activity during milling and extraction processes and cause severe trouble in soybean oil industry. Thermal inactivation of phospholipase D is a way to avoid non-hydratable phosphatides increase. Accurate determination of phospholipase D activity is a requisite tocontrol inactivation processes. Two methods were proposed to measure the phospholipase D activity. One method was based on the enzyme extraction which later acted on the substrate. The other method does not carry out the extraction of the enzyme, which remains in the original soybean matrix, acting directly on the substrate. High differences between phospholipase D activity values from thermallytreated soybeans were informed by applying both methods. Due such differences both methods were compared on the same thermally treated soybeans. The results showed that the values of the phospholipase D activity were lower when determined by the extraction method. This suggests that treatments might modify the enzyme extractability, and therefore, the activity values determined by the extractionmethod. According to these results, the method without extraction would be more appropriate for determining the phospholipase D activity.

Keywords: soybean, phospholipase D, enzymatic activity, thermal treatment, extraction method, direct-action method.
Introduction
Both the soybean seed and the crude oil contain soluble-in-oil phosphatides (Haraldsson, 1983). They might cause serious processingtrouble such as gum formation, oil darkening during deodorization and physical refining (Andersen, 1953) and catalyser poisoning during partial or total hydrogenation (Zhang et al. 1994), making phosphatides removal necessary. Phosphatides can be present as hydratable phosphatides, able to form gums with water, or as non-hydratable phosphatides (NHP), not able to form them. NHP concentrationvaries in the seed according to its ripening stage and environment conditions during harvest and storage. During the crunching and flaking stages hydratable phosphatides may become NHP due to phospholipase D (PLD). This enzyme catalyzes the hydrolysis of hydratable phosphatides, preferably the phosphatdyl-choline, the phosphatide with the highest concentration in soybean seed, turning it intophosphatidic acid, a NHP according to the reaction

|phosphatidyl-choline + H2O |PLD |phosphatic acid + choline [1] |
| |[pic] | |
| |Ca++ | |The most inexpensive procedure to remove phosphatides from crude oil is the aqueous degumming, which is carried out by adding water to form insoluble gums which are removed by centrifugation. Reaction [1] increases NHP content in crude oils, making its removal by means of aqueous degumming difficult.
Ong et al. (1981, 2001), List et al. (1990, 1992, 1993) and Tosi et al. (1999) among others,evidenced the importance of inactivating enzymes, especially PLD, in stages previous to solvent extraction. They concluded that thermal treatments can reduce NHP formation by inactivating PLD, thus allowing an inexpensive removal of phosphatides by aqueous degumming.
Determination of PLD activity (PLDA) is the key to apply thermal inhibition methods. Two different methods were used to determine...