Lipase activity in wheat ingredients A simple assay for lipase activity in wheat flour streams N. ZHOU (1), L. Haynes (1), W-K. Chung (1), L. Slade (2). (1) Kraft Foods, East Hanover, NJ, USA; (2) Food Polymer Science Consultancy, Morris Plains, NJ, USA. Introduction Lipase (EC 3.1.1.3) exists widely in nature, such as plants, animals, bacteria and molds. It hydrolyzes triglycerides into free fatty acids (FFA), which is an important biochemical reaction for life. For whole wheat flour, however, it reduces the flour shelf life due to the fast oxidation of FFA (Galliard, 1989, 1986; Vetrimani and Rao, 1990). Many efforts on developing whole grain products in the food industry in recent years, involve the inactivation of lipase to produce a stable whole grain flour. In order to monitor the stabilization process, a fast and simple assay to quantify the lipase activity is required. Many methods had been developed to analyze lipase activity (Frédéric Beisson, et al, 2000) for various purposes. There is no single universal lipase assay that applies to all situations. The choice of a particular method will depend on the specific requirements. The most reliable method that analyzes the true lipase activity uses triglycerides as substrate, and analyzes the free fatty acid (FFA) produced by the lipase. This, however, is time consuming and would require that test sample be free of FFA, which is not practical, especially in the food industry. Many methods use water soluble esters as substrates. A widely used substrate is para-Nitrophenyl Butyate (p-NPB). It has been linearly correlated to the rate of FFA released by lipase (r2=0.954), with a rate ratio of 0.54 of nitrophenol to FFA, indicating that p-NPB as a substrate is reliable in determining lipase activity (Blake et al, 1996). Others have indicated that it is useful in studies on the molecular dynamics of lipase (Tsujita et al, 1989). The objectives of this research were to: 1.) explore the possibility of using p-NPB as a substrate to analyze lipase activity in whole wheat flours and related mill streams such as bran, germ, endosperm, and mixtures of them at a given ratio; 2.) study the kinetics of wheat lipase to determine the maximum enzyme velocity (Vm) and the substrate concentration at half maximum velocity (Km) (Michaelis Menten constant); 3.) establish the assay conditions suitable to quantify lipase activity for the food industry that is simple, fast, and does not require complicated instrumentation. Abstact Whole grain flour tends to have a shorter shelf life, due to rancidity. Lipase has been shown to cause hydrolytic rancidity. It hydrolyzes triglycerides into free fatty acids and glycerol. Free fatty acids may have a soapy flavor, and are highly susceptible to oxidation and rancid flavor development. A spectrophotometric assay to quantitatively measure lipase activity in cereal products, specifically for wheat flours, was developed. The method uses p-nitrophenyl butyrate (p-NPB) as substrate. Lipase hydrolyzes p-NPB into p-nitrophenol (p-NP) and butyric acid. The absorption of p-NP was measured spectrophotometrically at 400 nm at pH 7.5. The kinetics of lipase were studied with a wheat germ lipase standard. The maximum reaction velocity (Vm) and Michaelis constant (Km) are 0.005891 A/min/0.5 mg and 0.1139 mM/0.5 mg, respectively. The method was used to evaluate lipase activity in a variety of wheat mill streams during whole grain milling. The results indicated that the wheat germ and bran contain significantly higher lipase activity than endosperm. This method is simple, easy, fast, and sensitive. It may be a useful tool for whole grain research and product development. Based on these results, the substrate concentration should be 2Km (2 X 0.1139 = 0.2278 mM), if lipase content is around 0.5 mg/ sample (activity of 5 microequivalent triacetin/hour at pH 7.4 at 37 C). If the reaction time is 30 min, substrate at concentration of 1.075 mM gives an absorbance in the range of 0.2 – 0.8. The lipase activity can be determined by measuring the change of the absorbance (DA) at a given reaction time within the initial linear range, or using a p-nitrophenol calibration curve to express its activity in moles of p-nitrophenyl hydrolyzed per unit time and weight. Substrate concentration should be above1 mM to ensure linear reaction kinetics. If the absorbance is above 0.8, the sample needs to be diluted. A simple procedure for whole wheat ingredients is recommended as follows, using the calibration curve on the right. Results Determine Vm and Km To analyze the enzyme activity accurately, its reaction with substrate needs to be in steady state. The selected time interval of the assay needs to be in the linear initial velocity stage, so that all enzyme is in the form of enzyme-substrate complex and the free substrate concentration vastly exceeds that of enzyme. The kinetics of the lipase reaction were observed with various substrate concentrations, the maximum reaction velocity (Vmax) and Michaelis Menten constant (Km) were determined, and the substrate concentration required to maintain steady state (>2Km) were calculated. Lipase activity in wheat ingredients Weigh samples (for wheat: flour – 0.05g, bran and germ – 0.02 g). Add 9 ml phosphate buffer (pH7.5). Add 1 ml of 10 mM p-NPB. Record the exact time of p-NPB addition. (The final concentration of p-NPB is 1 mM in the sample solution.) Shake the sample tube, leave it in 25 °C water bath. Centrifuge the sample at 1000 g for 5 min, 20 min after p-NPB addition. Measure the supernatant absorbance at 400nm at exactly 30 min after p-NPB addition. Use phosphate buffer (9ml) and p-NPB (1ml) mixture as blank, leave the blank in 25 °C water bath for 20 min, centrifuge, and measure the absorbance at 30 min at 400 nm. Calculate the sample absorbance by subtracting the blank absorbance from it. Calculate the lipase activity using the calibration curve. Figure on the right shows the change of cumulative absorbance with time at three different substrates levels (sample – blank). Data indicated that the reaction rate started to decrease (curve become nonlinear) around 50 minutes for lower levels of substrate (0.1 and 0.2 ml), but still in linear range for high level of substrate 1.0 ml). Principle: pNPB + lipase  p-nitrophenol + butyrate p-nitrophenol (pNP) has an aborption peak at 400nm Materials and Methods Materials: Wheat germ lipase: Sigma (St. Louis, MO), L-3001, 95% solid, 9.6 u/mg solid. One unit hydrolyzes 1.0 microequivalent triacetin/hour at pH 7.4 at 37 C; Para-Nitrophenyl butyrate: Sigma (St. Louis, MO), N9876, 98%; Phosphate buffer: pH 7.5, 0.2 M; Acetonitrile: Fisher Scientific (Fair Lawn, NJ), A998-4, HPLC grade; Instruments: Spectrophotometer; Centrifuge, capable of 1000g or higher; Water bath. Flour stream samples: Bran, germ, and white flour were obtained from a commercial mill. Procedures: Make p-NPB substrate solution (10.75 mM, 0.225g p-NPB in 100ml acetonitrile). For the kinetic study to determine Km and Vm: 0.5 ml lipase standard (1mg/ml) in phosphate buffer (pH7.5) was mixed with 0.1 to 1.0 ml of p-NPB substrate solution, additional phosphate buffer was added to reach the total volume of 10 ml. Absorbance at 400nm was measured starting 1 minute after mixing at least 3 hours. A substrate blank without lipase standard was run in the same way. For the sample measurement: 0.02 g bran/germ or 0.05 g white flour. Mix by shaking with 9 ml of phosphate buffer (pH7.5) and 1 ml p-NPB substrate (10.75mM). Incubate at 25 C for 20 minutes. Centrifuge at 1000 g for 5 minutes. Measure the supernatant absorbance at 400nm at exactly 30 minutes after the samples are mixed with substrate. A substrate blank was run without sample. rate The substrate concentration and linear reaction rates that were used to calculate 1/[S] and 1/V are listed in the following table. The table on the right lists the lipase activity of wheat bran, germ, shorts, and two break flours from a commercial mill, using the above procedure. The reaction rates between 5 – 30 min of all samples are in linear range. The rates are in the following figure. In this study, all samples had an absorbance at 400 nm of less than 0.05 in the absence of substrate, and the impact on the measured lipase activity was small and was ignored for practicality. If a sample has background absorbance more than 0.05, a sample blank without substrate should be subtracted. References Galliard, T. 1989. Rancidity in cereal products. In: Rancidity in foods. 2nd Ed. Allen, J., Hamilton, R. Elsevier Applied Science. New York. P141-146. Galliard, T. 1986. Hydrolytic and oxidative degradation of lipids during storage of wholemeal flour: effects of bran and germ components. J. Cereal Sci. 4:179-192. Vetrimani, R. and Rao, P. H. 1990. Studies on stabilization of wheat bran. J. Fd. Sci. Technol. 27:332-335. Beisson, F., Tiss, A.., Rivière, C., and Verger, R. 2000. Methods for lipase detection and assay: a critical review. Eur. J. Lipid Sci. Technol., 133–153. Tsujita, T. Ninomiya, H. and Okuda, H. 1989. p- NitrophenyI butyrate hydrolyzing activity of hormone-sensitive lipase from bovine adipose tissue. Journal of Lipid Research. 30:997-1004. Blake, M. R., Koka, R., and Weimer, B. C. 1996. A Semiautomated Reflectance Colorimetric Method for the Determination of Lipase Activity in Milk. J. Dairy Sci. 79:1164-1171. From the equation and above figure: Km = 0.1139 mM (per 0.5 mg lipase) Vm = 0.005891 A/min/0.5 mg lipase, or ~ 0.7 A/hour/mg lipase Based on the data in the table, the regression equation is calculated from 1/[S] and 1/V.