Determination of Lipoxidase in Wheat Germ

Lipoxidase in wheat germ affects the wheat germ’s quality and must be inactivated for processing. Because no method was available for measuring unpurified lipoxidase in wheat germ, methods for measuring purified lipoxidase or lipoxidase in soybean were used as reference methods. These methods proved to be so inaccurate that the natural state of the wheat germ could not be determined. A simple method was therefore established to determine lipoxidase in wheat germ. It includes enzyme extraction, substrate preparation, and conditions for termination of the enzymatic action. Furthermore, the optimum inactivation conditions for lipoxidase in wheat germ were tested. MATERIALS AND METHODS Materials and Instruments Fresh wheat germ was obtained from Shanghai Flour Company Ltd. Linoleic acid, with 99% purity, was Fluka-62230, and Tween 20 was Fluka-93773. The UV/ Vis spectrophotometer, model UV-2100PC, was obtained from UNICO (Shanghai) Instruments Co., Ltd. A Precisa electronic balance model XS225A, a homogenizer (model SilentCrusher) from S. Heidolph (Germany), and a centrifuge, model DL5B, from Shanghai Centrifuge Research Institute were used. Measurement of Lipoxidase in Wheat Germ Defatted wheat germ extract (0.3 mL) with proper concentration was added to 2 mL of boric acid buffer solution, pH 9.0, containing linoleic acid at 2.24 × 10 mol/ L. Both extract and buffer were kept at 30°C before mixing. The mixture was held for 4 min at 30°C, and then the enzymatic action was terminated by adding 2 mL of 1.5 mol/L NaOH. The optical absorption was detected at 234 nm. Definition of the Activity Unit of Lipoxidase in Wheat Germ In the above-mentioned lipoxidase measuring system, an increase of 0.001 in optical absorption caused by 1 g of wheat germ per minute was defined as 1 enzyme activity unit (U). RESULTS AND DISCUSSION Determination of lipoxidase activity in wheat germ is influenced by several factors: the defatting of the wheat germ, the RESEARCH extraction of lipoxidase, the concentration of the extract, and especially the method for stopping the lipoxidase reaction. Defatting of Wheat Germ If the wheat germ were not defatted, the linoleic acid existing in it would be catalyzed by lipoxidase during the extraction of lipoxidase, which would cause high optical absorption in the extract and make measurement of the enzyme activity impossible. The fats in wheat germ must be removed thoroughly, but enzyme activity must not be affected during the process. An appropriate method was found after many experiments. The wheat germ was crushed and filtered through a 100-mesh sieve. This wheat germ powder was then soaked in petroleum ether for 12–14 hr at 30°C. The amount of petroleum ether used was three times the wheat germ’s weight. The defatted wheat germ was filtered and evaporated at room temperature to remove residual petroleum ether. Extraction of Lipoxidase from Wheat Germ To extract lipoxidase from wheat germ, a suspension of 1 g of defatted wheat germ powder in water was shaken for 30 min at 30°C and centrifuged for 20 min at 5,000 rpm to a certain volume; this was used as the enzyme solution. The effect of concentration of the lipoxidase extract on the measurement of enzyme activity is shown in Table I, where increased enzyme activity correlates with increased absorption. When 1 g of enzyme extract was diluted to 10 mL, the enzyme reaction system had higher protein concentration and lower transparency, and the optical absorption value did not show an obvious change. When the enzyme extract was diluted to 50 or 100 mL, the optical absorption value changed relatively obviously. To analyze the activity of enzyme that had been partly inactivated by a heat treatment, the enzyme extract was diluted to 50 mL. Generally speaking, the addition of a homogenization step before shaking for 30 min was helpful for protein extraction. The effect of homogenization on the wheat germ extract’s lipoxidase activity is shown in Table II.