Effect of Starch on the Inactivation of Amylase in Starch-Containing Foods

Transcription

1 Food Sci. Technol. Res., 19 (6), , 2013 Note Effect of Starch on the Inactivation of Amylase in Starch-Containing Foods Kazuo Koyama 1*, Jinji Shono 1, Hiromu Taguchi 1, Akira Toriba 2 and Kazuichi Hayakawa 2 1 Central Research & Development Institute, House Foods Group Inc., 1-4 Takanodai, Yotsukaido-shi, Chiba , Japan 2 Graduate School of Natural Science and Technology, Institute of Medical Pharmaceutical and Health Science, Kanazawa University, Kakuma-machi, Kanazawa , Japan Received June 17, 2013; Accepted August 12, 2013 The quality of starch-containing foods is significantly impaired by contamination with small amounts of α-amylase, which hydrolyzes the starch and causes viscosity loss. We examined the effect of different temperatures and times on inactivation of α-amylase in starch-containing foods. Model foods containing a known amount of human salivary α-amylase (HSA) were incubated at temperatures from 60 to 80 for 30 min. In the case of a 3% starch suspension incubated at 70, it took 10 min before the viscosity loss ceased, by which time the viscosity was halved. In 0.1 3% starch suspensions at ph 5.5, the inactivation of HSA could be described by a first order kinetic model. The presence of starch decreased the inactivation rate constant by decreasing the Arrhenius frequency factor. To prevent viscosity loss by α-amylase contamination in starch-containing foods, the inactivation time and temperature should be increased with increasing concentration of starch. Keywords: α-amylase, inactivation, thermostability, starch-containing food, viscosity *To whom correspondence should be addressed. k-koyama@housefoods.co.jp Introduction Effective use of starch in the food industry requires a thorough understanding of the enzymatic properties of amylase and the degradation behavior of starch. The increase in viscosity of starch-containing foods is largely due to the gelatinization of starch during heating. Viscosity is an important quality factor for thickened foods, such as soups, stews and curries that contain 5% or less starch. If a trace amount of amylase contaminates starch-containing foods, it can hydrolyze the starch and cause viscosity loss. Possible sources of amylase contamination include honey, spices, grains, fermented food, human saliva left on a tasting spoon and enzyme-containing dishwashing detergent. To prevent viscosity loss by non-thermophilic amylase contamination, starch-containing foods need to be heated to inactivate the amylase. However, using more heat than is necessary will reduce the freshness of the food. Food manufacturers have empirically found that heating at approx. 80 results in amylase inactivation. The thermostability of amylase increases in the presence of polyols, sugar and starch. Busch and Stutzenberger (1997) found that the addition of 2 5% soluble starch (an amount similar to that in starch-containing foods) delayed the decrease in activity of thermophilic α-amylase incubated at 70. However, they did not examine the inactivation rate constant or the time course of viscosity loss by amylase. Adding 50% starch reduced the inactivation rate of hyperthermophilic α-amylase, allowing the enzyme to be stable at 108 (de Cordt et al., 1994). The enzyme stabilization can be explained by that the protein is preferentially hydrated by the addition of reagent. Little is known about the effect of low concentrations of starch on non-thermophilic α-amylase at approx. 80. Although α-amylase can decrease the viscosity of a starch suspension, changes in α-amylase activity require elucidation. Liquid sugar is produced from starch by the addition of excess thermophilic α-amylase, but changes in activity were not monitored. Honey or saliva contamination was found to reduce the viscosity of a starch suspension (Babacan et al., 2002; Hanson et al., 2012), although these studies did not measure the time course of amylase activity. We investigated the relationship between the inactivation of non-thermophilic amylase at temperatures of 60 80

2 990 and the viscosity of starch-containing foods. In this study we used human salivary α-amylase (HSA) because it is one of the most common contaminants in cooking. Our results show that the viscosity loss can be explained by the time course of α-amylase inactivation. Moreover, the presence of starch increases the thermostability of α-amylase. Materials and Methods Reagents HSA was purchased from AppliChem GmbH (Darmstadt, Germany). Wheat starch, NaCl, tris (hydroxymethyl) aminomethane, HCl, NaOH, citric acid, sodium phosphate, ammonium acetate, acetonitrile, and iodine solution were purchased from Wako Pure Chemical Industries (Osaka, Japan). 3-Morpholinopropanesulfonic acid (MOPS) was purchased from Dojindo Laboratories (Kumamoto, Japan). As the fluorescent substrate, dye quenched starch (DQ starch) labeled with BODIPY was obtained from Life Technologies (NY, USA). Bovine serum albumin (BSA) was purchased from Sigma-Aldrich Japan (Tokyo, Japan). Curry roux was purchased from a local market. α-amylase standard Two series of α-amylase solutions were prepared. The α-amylase solutions ( U/mL) used for spiking model foods were prepared by dissolving the α-amylase (Bacillus subtilis, EC , 111 U/mg solid) in 100 mm MOPS buffer, ph 6.9. The α-amylase solutions used for constructing a calibration curve ( mu/ml) were prepared by dissolving the α-amylase in 20 mm tris-hcl buffer containing 0.2 M NaCl, ph 8.5. Model foods Four types of model foods were prepared for α-amylase addition. Food products typically have a low ph and contain salt to ensure bacteriostasis as well as to meet consumer demand for saltiness. A 20 mm citrate phosphate buffer at ph 5.5 was used as the buffer for model foods. Model starch suspensions were prepared by mixing % (w/w) starch and 1.1% (w/w) NaCl with buffer, and boiling for 5 min to thicken the slurry by gelatinization. A model curry sauce was prepared by boiling an aqueous solution of 10% (w/w) commercial curry roux (containing starch and NaCl, ph 5.5) for 5 min. Buffer containing 1.1% (w/w) NaCl was used as the reference solution. Model foods were incubated at 60, 70 and 80 in a thermostatic water bath. The reference solution was incubated at 50, 60 and 70. Time course study of viscosity loss by α-amylase The model foods were pre-heated by placing the measuring vessels in a thermostatic water bath. HSA and BSA were added to 15 ml of food models to obtain test samples containing 30 mu/ml of HSA and 0.1% (v/v) BSA. Test samples were immediately placed in the water bath at 60, 70 or 80. The heating time was 0 to 30 min. Viscosity was measured with a viscometer DVL-B2 equipped with HM-2 rotor (Toki K. Koyama et al. Sangyo, Tokyo, Japan) at 60 rpm. Thermostability study of α-amylase The model foods were pre-heated by placing the tubes in a thermostatic water bath. HSA and BSA were added to 2 ml of model foods to obtain test samples containing 30 mu/ml of HSA and 0.1% BSA. Test samples were immediately placed in a water bath at 60, 70 or 80, and heated for 0 to 30 min. Aliquots were removed at designated time intervals and immediately transferred to ice and applied to the amylase assay. The α-amylase activity in model foods was measured by modifying our previous method (Koyama et al., 2012). The principle of this method is based on monitoring the fluorescent substrate digested by α-amylase, combined with steps to avoid interference caused by starch, dye and impurities in food samples. To remove insoluble starch and contaminants from the model foods, the test samples were centrifuged at 20,000 g for 10 min. The supernatant was filtered through No. 5B filter paper and used as the crude extract. Pre-purification steps were conducted to exclude competitive inhibition by starch derivatives present in the crude extract. The crude extract (1.0 ml) was desalted by applying it to a PD-10 column (8.3 ml gel, GE Healthcare UK, Buckinghamshire, UK) equilibrated with 20 mm Tris-HCl, ph 8.5 (buffer A). The desalted amylase was eluted with 4.5 ml of buffer A and loaded on a DE- AE-Toyopearl 650M (anion-exchange) column (1.5 ml gel, Tosoh, Tokyo, Japan) equilibrated with buffer A. After the column was washed with 6.0 ml of buffer A, the α-amylase was eluted using 4.0 ml of 0.2 M NaCl in buffer A (buffer B). The purified α-amylase solution (50 μl) was added to 50 μl of 200 μg/ml BODIPY-labeled DQ starch dissolved in 100 mm MOPS buffer, ph 6.9, and the mixture was incubated for 30 min at room temperature in the dark. A calibration curve was created by using 50 μl of blank sample and 0.75, 2.5, 7.5 and 12.5 mu/ml α-amylase solutions (corresponding to test samples containing 3, 10, 30 and 50 mu/ml). Acetonitrile (100 μl) was immediately added to the reaction mixture and mixed to inactivate the α-amylase. The mixture was centrifuged at 13,800 g for 10 min. The supernatant was filtered with a 0.20 μm membrane. The fluorescence of the test solution was measured by HPLC (Agilent Technologies, CA, USA). The fluorescent substrate fraction was separated on an L-column2 C18 (2.1 mm 150 mm, 3 μm; CERI, Saitama, Japan), with an L-column2 C18 (2.1 mm 50 mm, 5 μm) as guard column. Mobile phases A and B were 20 mm ammonium acetate aqueous solution and acetonitrile, respectively. The gradient was as follows: 10 25% (v/v) B from 0 to 7 min, 25 65% B from 7 to 17 min, 65 95% B from 17 to 20 min, 95 10% B from 20 to 29 min (a total run time of 40 min). The flow

3 Effect of Starch on Amylase Stability rate was 0.2 ml/min and the column temperature was kept at 40. The fluorescence wavelength of the detector was set at Ex / Em = 502 / 512 nm. Analysis of thermostability data Kinetic parameters were calculated from heating experiment data according to the method of Ludikhuyze et al. (1996). The first-order rate inactivation rate constants (k, min 1 ) were calculated from logarithmic activity data as a function of heating time (t, min). A t = k 0 exp( kt) (1) where A 0 is the initial enzyme activity and A t is the activity at time t. Using Eq. (1), the inactivation rate constant for different temperatures can be calculated by linear regression. The frequency factor (k 0 ) and activation energy (E a ) are derived from the regression of ln k versus (1/T) using the Arrhenius Eq. (2). E a k = k 0expd- n RT (2) where R is the gas constant, and T is the absolute temperature. Results and Discussion α-amylase activity at high temperatures Fig. 1 shows the time course of the viscosity of starch suspensions spiked with 30 mu/ml of HSA at 60, 70 and 80. The temperatures were selected to mimic viscosity loss that occurs by saliva contamination during cooking. Relative viscosity was defined as the ratio of the viscosity of the starch suspension spiked with HSA to the pure starch suspension. At 60, viscosity was rapidly reduced and was almost lost within 10 min. At 70, viscosity was reduced by half within 10 min and remained unchanged until 30 min. At 80, viscosity remained almost completely unchanged. Bacterial amylase was found to cause viscosity loss in an oatmeal suspension (Jansson and Lindahl, 1991) and in wheat starch (Leman et al., 2006). Saliva contamination was found to reduce the viscosity of a maize starch-thickened drink used in clinical management of dysphagia (Hanson et al., 2012). These studies did not assess amylase activity or investigate the conditions needed to inactivate amylase in starch suspensions. Honey amylase was found to cause viscosity loss in a waxy maize suspension at 40 (Babacan et al., 2002; Babacan and Rand, 2007). They reported that the amylase could be inactivated by heating the honey to 83. However, the relationship between amylase activity and viscosity in starch suspensions remains unknown. Fig. 2 shows the inactivation plot of 30 mu/ml of HSA purified from the 3% starch suspensions used at 60, 70 and 80 for 30 min in the viscosity experiment (Fig. 1). Inacti- Relative viscosity (%) Fig. 1. Time course of viscosity loss of 3% (w/w) starch suspensions by human salivary α-amylase. Starch suspensions were spiked with 30 mu/ml α-amylase and incubated for 30 min at 60, 70 and 80. Relative viscosity was determined as the ratio of the viscosity of the spiked sample to the viscosity of the control sample o C 80 o C 65 o C Fig. 2. Inactivation of human salivary α-amylase in 3% (w/w) starch suspension at 60, 65, 70 and 80. A t ; enzyme activity at time t, A 0 ; enzyme activity at t = 0. vation is a first-order process, hence, for each temperature, an inactivation rate constant (k) was calculated. The ratio of the activity at time t to the initial activity, A t /A 0, remained close to 1 at 60, dropped to about 0.1 (an activity loss of about 90%) after 15 min at 70, and dropped to 0.01 (an activity loss of about 99%) within 1 min at 80. As shown in Fig 1, the relative viscosity at 70 decreased for about 15 min and then stopped decreasing when the residual activity would be about 10 1 of the initial activity (A t /A 0 = 0.1). At 80, little or no loss of relative viscosity was observed because of the rapid inactivation of the enzyme. Effect of starch on the stability of α-amylase Fig. 3 shows the time course of thermal inactivation of HSA in

4 992 the reference solution (without starch) at 50, 60 and 70. α-amylase activity was maintained for 30 min at 50, but decreased to A t /A 0 = 0.1 after 10 min at 60 and within 1 min at 70. A comparison of Figs. 2 and 3 suggests that the inactivation of HSA in the presence of 3% starch requires a higher temperature than the inactivation in the absence of starch. Low concentrations of starch enhanced the thermostability of α-amylase. Fig. 4 shows the residual activity of 30 mu/ml HSA purified from different model foods heated for various time at 70. The activity of the enzyme was reduced to 10 1 of the initial activity in 1 min in the reference solution (without starch), in 5 min in 0.1% starch suspension, and in 15 min in 3% starch suspension, indicating that the starch, even at low concentrations, enhanced the thermal stability of HSA. The thermal stability of HSA in curry sauce, which contains 3% starch, was about the same as that of the 3% starch suspension. Two other food components, 3.6% lipid and 0.6% protein, in the curry sauce appeared to have negligible effects on the thermostability of HSA. For model foods, inactivation rate constants (k) of HSA are calculated from graphs of residual activity versus heating time (e.g., Fig. 2). The inactivation rate constants were plotted against the reciprocal of the absolute temperature to make Arrhenius plots (Fig. 5), and frequency factors (k 0 ) and activation energies (E a ) determined from the regression are shown in Table 1. The k 0 and E a values of the enzyme decreased with the increase in starch concentration. The presence of starch reduced the HSA inactivation rate predominantly by decreasing the k 0. The effect of E a on the inactivation rate was small.other agents have been shown to have stabilizing effects on different types of amylases. Amylase can be stabilized by either (i) a high concentration of polyols, starch and sugar, or (ii) a low concentration of starch. (i) A protein in aqueous solution is stabilized by preferential interactions, i.e., the hydration of a protein increases with increasing concentration of an additional agent (Arakawa and Timasheff, 1982). Adding 15% glycerol reduced the inactivation rate, i.e., enhanced the thermostability, of Bacillus subtilis α-amylase (Ludikhuyze et al., 1996), while the addition of 50% starch reduced the inactivation rate of α-amylase from the hyperthermophilic Bacillus licheniformis, allowing the enzyme to be stable at 108 (de Cordt et al., 1994). In both studies, the reaction of starch hydrolysis by α-amylase was not affected by an additional agent (i.e., not affected by preferential interaction) because the amylase did not reduce E a values. We assume that the amylase was in the form of a paste or solid in the presence of excessive agent, and did bind to starch as described in the following section. 50 o C K. Koyama et al Fig. 3. Inactivation of human salivary α-amylase in a reference solution (20 mm citrate-phosphate buffer, ph 5.5, containing 1.1% (w/ w) NaCl) at 50, 60 and 70. Reference solution 0.1% Starch Curry 3% Starch Fig. 4. Inactivation of human salivary α-amylase in different model foods at 70. k (min -1 ) 3% Starch Curry 0.1% Starch Reference solution /T (K -1 ) Fig. 5. Arrhenius plots of human salivary α-amylase in different model foods. Inactivation rate constants (k) were calculated from logarithmic activity data as a function of heating time (e.g., Fig. 2).

5 Effect of Starch on Amylase Stability Table 1. Frequency factors (k 0 ) and activation energies (E a ) for various model foods, derived from regression of log (k) versus (1/T) from Fig. 5. Model food k 0 (min 1 ) E a (kj/mol) Reference model solution % Starch suspension % Starch suspension Curry sauce (ii) In studies in which low concentrations of starch were added, the thermostability of amylase was expressed as a decrease in activity with time. The addition of 2 5% soluble starch delayed the decrease in the activity of Thermomonospora fusca α-amylase incubated at 70 (Busch and Stutzenberger, 1997) and delayed the decrease in the activity of thermophilic Bacillus sp. WN11 incubated at 105 (Mamo et al., 1999). These studies concluded that starch had a stabilizing effect on amylase, although they did not measure k 0 and E a values. The previous reports (i and ii) still measured high amylase activities (1 U/mL or more) by using conventional methods. However, for quality control, viscosity loss of liquid starch-containing food can be significant at amylase concentrations less than 0.1 U/mL. Our results show that starch increases the stability and the reaction rate of a trace amount of α-amylase. The k 0 and E a of HSA decreased in the presence of starch (Table 1), suggesting that HSA binds starch, forming an enzyme-substrate complex that stabilizes the enzyme. Enzyme-substrate complex formation requires a substrate concentration corresponding to the K m value. The K m value of HSA with maltotetraose as substrate is 0.2% (Minaminura, 1988). We assume the K m value of HSA with starch is 0.2% or less because the K m value of microbial α-amylase with soluble starch (0.053%) is smaller than that with maltotetraose (0.55%) (Ohno et al., 1992). Other studies reported that the K m values of microbial α-amylase were less than 0.8% (Gupta et al., 2003; Busch and Stutzenberger, 1997). The present results suggest that HSA formed a complex with starch when in the presence of 0.1 3% starch suspensions serving as model foods. Conclusions We investigated the inactivation rate of human salivary α-amylase in model foods heated to temperatures between 60 and 80. The presence of starch was found to decrease inactivation rate constants of α-amylase by decreasing the Arrhenius frequency factor. These results suggest that HSA binds starch, forming an enzyme-substrate complex that stabilizes the enzyme. To prevent viscosity loss by α-amylase 993 contamination in starch-containing foods, it is necessary that heating temperature and time should be increased to inactivate the α-amylase depending on the concentration of starch. This work will facilitate the design of liquid starch-containing foods in which viscosity is maintained. References Arakawa, T. and Timasheff, S.N. (1982). The stabilization of proteins by osmolytes. Biochemistry, 21, Babacan, S., Pivarnik, L.F. and Rand, A.G. (2002). Honey amylase activity and food starch degradation. J. Food Sci., 67, Babacan, S. and Rand, A.G. (2007). Characterization of honey amylase. J. Food Sci., 72, C050-C055. Busch, J.E. and Stutzenberger, F.J. (1997). Amylolytic activity of Thermomonospora fusca. World J. Microbiol. Biotechnol., 13, de Cordt, S., Hendrickx, M., Maesmans, G. and Tobback, P. (1994). The influence of polyalcohols and carbohydrates on the thermostability of α-amylase. Biotechnol. Bioeng., 43, Gupta, R., Gigras, P., Mohapatra, H., Goswami, V.K. and Chauhan, B. (2003). Microbial α-amylases: a biotechnological perspective. Process. Biochem., 38, Hanson, B., O Leary, M.T. and Smith, C.H. (2012). The effect of saliva on the viscosity of thickened drinks. Dysphagia, 27, Jansson, K.W. and Lindahl, L. (1991). Rheological changes in oatmeal suspensions during heat treatment. J. Food Sci., 56, Koyama, K., Hirao, T., Toriba, A. and Hayakawa, K. (2012). An analytical method for measuring α-amylase activity in starchcontaining foods. Biomed. Chromatogr., 27, Leman, P., Bijttebier, A., Goesaert, H., Vandeputte, G.E. and Delcour, J.A. (2006). Influence of amylases on the rheological and molecular properties of partially damaged wheat starch. J. Sci. Food Agric., 86, Ludikhuyze, L., de Cordt, S., Weemaes, C., Hendrickx, M. and Tobback, P. (1996). Kinetics for heat and pressure-temperature inactivation of Bacillus subtilis α-amylase. Food Biotechnol., 10, Mamo, G., Gashe, B.A. and Gessesse, A. (1999). A highly thermostable amylase from a newly isolated thermophilic Bacillus sp. WN11. J. Appl. Microbiol., 86, Minaminura, N. (1988). Handbook of amylases and related enzymes. The amylase research society of Japan, ed. by Pergamon Press, pp Ohno, N., Ijuin, T., Song, S., Uchiyama, S., Shinoyama, H., Ando, A. and Fujii, T. (1992). Purification and properties of amylases extracellularly produced by an imperfect fungus, Fusidium sp. BX-1 in a Glycerol Medium. Biosci. Biotechnol. Biochem., 56,