Heat-Moisture Treatments of Cowpea Flour and Their Effects on Phytase Inactivation NICOLE S

JFS E: Food Engineering and Physical Properties

Heat-moisture Treatments of Cowpea Flour and Their Effects on Phytase Inactivation NICOLE S. AFFRIFAH, MANJEET S. CHINNAN, AND R. DIXON PHILLIPS

ABSTRACT: Samples of finely ground cowpea flour with moisture content adjusted to 10%, 25%, 35% (dry basis) were heated in sealed retort pouches at 70 to 95 °C for periods of 2 to 32 min. Phytase showed a high thermal resistance with residual activity ranging between 50% and 95%. Thermal inactivation of cowpea phytase was adequately described by a fractional conversion model based on a 1st-order rate equation. Overall, increasing temperature and initial moisture content resulted in increased enzyme inactivation. Estimated activation energies between 70 and 95 °C were 33.3, 37.9, and 43.4 kJ/mol at 10%, 25%, and 35% moisture, respectively. The kinetic models generated were successfully used to predict phytase activity in cowpea flour. Keywords: phytase, cowpeas, inactivation kinetics, moisture content, fractional conversion

Introduction 1998). The enzyme is widely distributed both in plants and animals owpeas are an important source of protein in the diets of many as well as various fungi and bacteria species (Cosgrove 1966).

E: Food Engineering & Physical Properties Cpopulations around the world providing a less expensive al- Phytases are generally thermostable enzymes with pH optima ternative to animal protein. Despite the high nutritional quality of between 4.0 and 5.6 (Dvorakova 1998). Although Jongbloed and cowpeas and other legumes, limitations such as the hard-to-cook Kemme (1990) previously showed that phytase was readily inacti- defect decrease consumption and production. The basis of the de- vated at temperatures of 70 °C or higher, Ma and Shan (2002) re- fect is still under debate; however, a dual-enzyme mechanism in- ported a high thermal stability in 3 cereal seeds. After heating for volving phytase and pectin methylesterase has been suggested 1 h at 100 °C, rye2, wheat NEAU123, and triticale 5305 retained (Jones and Boulter 1983). It is hypothesized that phytase acts on more than 80% of their original phytase activity. phytic acid in the cotyledon releasing inorganic phosphate, mag- Based on the reported relationship between phytase activity and nesium, and calcium ions while pectin methylesterase hydrolyz- cooking time of various legumes, we are hypothesizing that inac- es pectin to pectinic acid and methanol in the middle lamella tivating phytase could be used as a strategy in controlling the hard- (Mafuleka and others 1993). The calcium and magnesium ions to-cook defect. There is however a scarcity of information on the move to the middle lamella forming calcium and magnesium pec- behavior of phytase following heat treatment. Thus understanding tates that subsequently restrict cell separation during cooking. the inactivation kinetics would help in designing heat processes Several studies have examined the relationship between the targeted at preventing or reducing the development of the hard- development of the hard-to-cook defect and phytate content. Kon to-cook defect in legumes. The objectives of this study were to de- and Sanshuck (1981) reported a negative correlation between the termine the effectiveness of heat treatments in inactivating phytase phytic acid/calcium ratios and cooking time in dry beans. A low but in cowpeas and to model and estimate the kinetics of thermal in- significant correlation (r = –0.716) was observed between losses in activation as a function of moisture content. phytate and cooking time in black beans suggesting that phytate is a contributor, but perhaps not the sole operating mechanism, in Materials and Methods the hardening defect (Hincks and Stanley 1986). Longe (1983) also reshly harvested cowpea seeds were obtained from SeedGrow, reported that phytic acid was the only chemical component among FLLC (Meridian, Calif., U.S.A.) and stored at 4 °C until used. All 4 others that showed a correlation with the cooking time of 13 va- chemicals used were of analytical grade and obtained from Sigma- rieties of cowpeas. Mafuleka and others (1993) recorded increased Aldrich Chemical Co. (St. Louis, Mo., U.S.A). phytase activity and a strong positive correlation with cooked tex- ture in decorticated Malawian red and white bean genotypes stored Conditioning of cowpea flour under different temperature, water activity, and time periods. Cowpea seeds were finely ground in a Wiley laboratory mill (Model Phytases are a subfamily of high molecular weight histidine acid 4, Arthur H. Thomas Co. Philadelphia, Penn., U.S.A.) to pass through phosphatases that catalyze the hydrolysis of phytic acid to inosi- a nr 40 mesh sieve. The moisture content of the flour was adjusted by tol and free orthophosphate (Liu and others 1998; Viveros and oth- adding a pre-determined amount of water to the flour in a mixer. The ers 2000). The main end products of the reaction are phosphoric amount of water required was calculated using the following equation: acid and myo-inositol with intermediate compounds such as phos- photidyl inositols representing various degrees of dephosphoryla- (1) tion from inositol hexakisphosphate to inositol (Liu and others

MS 20040459. Submitted 7/7/04, Revised 8/18/04, Accepted 10/18/04. Authors where W is amount of water required (g/100 g), A is initial moisture are with Dept. of Food Science and Technology, Univ. of Georgia, 1109 Ex- content (g/100 g), B is dry matter in original sample (g/100 g), C is periment St., Griffin, GA 30223. Direct inquiries to author Affrifah (E-mail: final moisture content (g/100 g), and D is dry matter in final sample [email protected]). (g/100 g).

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The experiments were performed at moisture contents of 10%, ic model and the inactivation rate constant (k) can be estimated us- 25%, and 35% (dry basis). About 300 g flour was conditioned at each ing linear regression analysis (Wiley 1994; Whitaker 1994). moisture content, sealed in high density polyethylene bags, and

stored for 24 h at 4 °C to allow for equilibration. The sealed bags At = Ao exp (–kt) (2) were kept at 4 °C until their use within a period of about 7 d. Mois-

ture content was determined by a standard AOAC method (AOAC where Ao and At are the initial activity and residual activity at time 1980). t, respectively. Ly-Nguyen and others (2002) described a special case of the 1st- Thermal inactivation experiments order model known as the fractional conversion model that was A 6 ϫ 5 factorial design with 6 levels of heating temperature (70 applied in this study. Their article provides an outline of the basis to 95 °C) and 5 levels of heating time (2 to 32 min) was used. Retort and details of the model, as shown in the following summary. Frac- pouches (15.5 cm ϫ 6.5 cm) obtained from CLP Packaging Solu- tional conversion (f) accounts for the nonzero activity (Aϱ) after tions Inc. (Fairfield, N.J., U.S.A.) were used for the thermal inacti- prolonged heating and can be expressed as vation study. About 3 g flour at the desired moisture content was thinly dispersed in the pouch to a thickness of 3.6 mm and vacu- um sealed. The pouches were heated in an ethylene glycol bath (3) with controlled temperature (Tdesired ± 3 °C). Thermal inactivation was carried out at heating temperatures ranging from 70 to 95 °C and exposure times between 2 to 32 min. The temperature in the For most irreversible 1st-order reactions, Aϱ approaches zero, pouch was estimated by inserting a 30 gauge k-type thermocouple and Eq. 3 can be reduced to with a 0.01-inch bead (Omega Engineering, Stamford, Conn., U.S.A.) in a similar pouch and recording the change in temperature over the heating time. The come-up time, which was the time re- (4) quired to reach 99% of the desired temperature, was between 10 and 20 s depending on the final temperature desired. The pouches A plot of the logarithm of (1-f) against time results in a straight were removed from the heating bath as a function of the inactiva- line with a rate constant expressed by the negative slope value: tion time and were immediately cooled in a water-ice mixture for 5 min. The residual phytase activity in the flour sample was determined soon after using the method described below. A control (5) sample that was not subjected to any heat treatment was includ- ed for comparison. The phytase activity in this control sample was Thus, when Aϱ approaches zero, Eq. 5 and 2 are identical. considered as the initial phytase activity (Ao). Thermal treatments of the flour were performed in triplicate. To account for the nonzero activity after prolonged heating, the following form of the fractional conversion is employed: Phytase activity assay The phytase activity was measured by direct incubation with (6) sodium acetate buffer according to the method described by Grein- er and Egli (2003) with some slight modifications. The heat-treated E: Food Engineering & Physical Properties sample (1 g) was suspended in 20 mL of 0.1 M sodium acetate buff- Rearranging Eq. 6 yields Eq. 7: er, pH 5.0, containing 100 ␮mol of sodium phytate preincubated

at 45 °C. The reaction mixture was incubated at 45 °C for 30 min At = Aϱ + (A0 – Aϱ) exp(–kt) (7) after which the amount of phosphorus released was determined

using the method described by Eeckhout and De Paepe (1994). A By plotting At against inactivation time at constant temperature 2-mL portion of the reaction mixture was added to a test tube con- conditions, the inactivation rate constant, k, and the remaining taining 2 mL of 10% trichloroacetic acid to arrest the reaction and activity, Aϱ, can be estimated using nonlinear regression analysis. then centrifuged at 10000 ϫ g for 5 min. One milliliter of the super- The nonlinear estimation function in STATISTICA (StatSoft, Tulsa, natant was then added to 1 mL of a color-forming reagent. The col- Okla., U.S.A.) was used with the sum of squares of the relative er- or reagent was a mixture of 4 parts of solution A (15 g ammonium ror between measured and calculated enzyme activity used as the heptamolybdate in 55 mL 36 N H2SO4, made up to 1 L) and 1 part optimization criterion. of solution B (27 g of FeSO4.7H2O, a few drops of 36 N H2SO4, made The rate constants determined above were replotted in Arrhe- up to 250 mL). The blue color formed was measured at 700 nm in nius plots at the moisture contents studied and the activation

a diode array spectrophotometer (Model 8451, Hewlett Packard, energy (Ea) calculated from the slope according to Eq. 8 (Anthon Palo Alto, Calif., U.S.A.) after centrifuging at 10000 ϫ g for 3 min to and Barrett 2002). remove any cloudiness formed. A calibration curve was produced over the range of 1 to 4 ␮mol of phosphate and used to estimate the enzyme activity. The analysis was performed in triplicate and re- (8) ported as units/kg flour. One unit of phytase activity was defined as the amount of phytase that liberates inorganic phosphorus from ␮ a 0.001-M Na-phytate solution at a rate of 1 mol/min at pH 5 and Results and Discussion 45 °C. hytase proved to be fairly heat stable. The enzyme retained al- most 63% of its initial activity after subjection to the highest Kinetic data analysis P temperature (95 °C) and time (32 min) combination at the lowest Inactivation of enzymes can be described by a 1st-order kinet-

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moisture content (10%). The reduction in activity was determined Table 1—ANOVA summary table for phytase inactivation to a large extent by the moisture content of the flour prior to heat Factor DF F-ratio treatment. Overall, the reduction in phytase activity relative to ini- Moisture 2 90515.20a tial activity ranged between 5% to 37% for samples heated at 10% Temperature (Temp) 5 13065.00a moisture content whereas heating at 25% and 35% moisture result- Time 4 2733.65a ed in 20% to 56% and 29% to 64% reduction in phytase activity re- Moisture ϫ Temp 10 440.75a ϫ a spectively. Other studies have also reported that plant phytase (dry Temp Time 20 8.01 Moisture ϫ Time 8 105.96a flour) showed a high heat resistance. According to Ma and Shan Moisture ϫ Temp ϫ Time 40 26.76a (2002), wheat and rye phytase retained 89.47% and 104.64%, re- aSignificant at P < 0.01 spectively, of their activity after heating at 100 °C for 1 h. All factors considered in the present study (moisture content, temperature, and time) had significant effects on the residual phytase activity fol- increasing temperature and moisture content (Table 2). The high lowing the heat treatments (Table 1). Increasing temperature has values obtained are indicative of the high thermal stability of the a general effect of increasing enzyme activity up to an optimum af- phytase enzyme. The variation in Aϱ values implies that the suscep- ter which the activity decreases as the enzyme is progressively in- tibility of the stable enzyme fraction to heat inactivation is depen- activated (Parkin 1993). It was therefore expected that residual dent on treatment combinations. This further suggests that under phytase activity would decrease with increasing temperature. Ad- the appropriate conditions of heating temperature and moisture ditionally, the sensitivity of an enzyme to thermal inactivation is content, it might be possible to completely inactivate the enzyme dependent on the water content of the environment such that enzymes in dry or semi-moist food systems tend to be more heat stable (Parkin 1993). This is because water is needed to promote the unfolding of proteins during thermal denaturation. A plot of residual phytase activity as a function of heating time 85 10% resulted in nonlinear curves irrespective of heating temperature or initial moisture content (Figure 1). The thermal inactivation of E: Food Engineering & Physical Properties 70 phytase in cowpea flour could not be adequately described by a simple 1st-order kinetic model. Generally, the plots showed an initial drop in phytase activity during the 1st few min of heating at 55 each temperature. Beyond this, there appeared to be a gradual lev- eling in activity as the residual activity apparently stabilized. The 40

results suggest that the decrease in phytase activity may be more phytase activity (units/kg) dependent on heating temperature with the effect of heating time not being as pronounced especially for samples treated at high 25 temperatures (80 to 95 °C). None of the treatment combinations 0 4 8 121620242832 studied resulted in complete inactivation of the enzyme. Although Heating time (min) it is not known if isozymes of cowpea phytase exist, multiple en- 85 25% zymes have been reported for some plant seeds including lettuce

(Shannon 1968), lupine (Greiner 2002), and soybean (Hamada 70 1996). The use of whole flour rather than a purified enzyme fraction could have introduced isozymes that could account for the de- 55 viation from simple 1st-order kinetics. It has also been reported that there is an association between phytase and a non-specific acid phosphatase also capable of hydrolyzing phytic acid (Lolas and 40 Markakis 1977). phytase activity (units/kg)

The data were adequately described by a fractional conversion 25 model in the temperature range studied (70 to 95 °C). This suggests 0 4 8 121620242832

the presence of a stable enzyme fraction that was apparently not Heating time (min) significantly affected by the processing conditions under study. This 85 behavior was observed at all the 3 moisture contents studied. The 35% fitting of this model to the experimental data is shown in Figure 1. 70 The fractional conversion model was used to estimate the kinetic

inactivation parameters: namely, the initial phytase activity (Ao), the inactivation rate constant (k), and the amount of enzyme remain- 55 ing after treatment (Aϱ). These parameters were estimated by fit-

ting individual data points to the model using nonlinear regression 40

and are summarized in Table 2. phytase activity(units/kg) The initial phytase activity estimated using nonlinear regression was found to be between 81.4 and 84.0 units/kg. This was compa- 25 rable to the average initial activity (82.3 units/kg) measured in the samples. The fraction remaining after the longest heating time varied with the heating temperature and initial moisture content. Figure 1—Thermal inactivation of phytase in cowpea flour as The values ranged between 32 and 72 units/kg representing ap- a function of heating time at various temperatures and mois- proximately 38% and 88% of the initial activity, and decreased with tures

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Table 2—Kinetic parameters for phytase inactivation in cowpea flour Moisture content –1 2 dry basis) Temp. (°C) Ao (units/kg) Aؕ (units/kg) k (min ) R ,%) 10 70 81.95 ± 0.19a 72.66 ± 0.17 0.169 ± 0.012 0.937 75 82.24 ± 0.15 71.80 ± 0.08 0.229 ± 0.007 0.978 80 81.56 ± 0.19 68.60 ± 0.17 0.237 ± 0.029 0.815 85 81.78 ± 0.37 65.68 ± 0.37 0.240 ± 0.019 0.877 90 81.97 ± 0.22 60.24 ± 0.27 0.345 ± 0.016 0.951 95 82.22 ± 0.19 57.03 ± 0.39 0.407 ± 0.016 0.926 25 70 81.84 ± 0.18 58.96 ± 0.20 0.421 ± 0.024 0.938 75 83.06 ± 0.24 56.43 ± 0.29 0.466 ± 0.023 0.934 80 81.43 ± 0.17 46.76 ± 0.19 0.604 ± 0.011 0.975 85 81.53 ± 0.17 42.89 ± 0.25 0.663 ± 0.032 0.951 90 83.98 ± 0.17 39.90 ± 0.23 0.782 ± 0.021 0.966 95 82.32 ± 0.17 38.65 ± 0.16 1.104 ± 0.023 0.981 35 70 82.18 ± 0.17 51.67 ± 0.16 0.678 ± 0.022 0.986 75 82.32 ± 0.16 47.20 ± 0.12 1.136 ± 0.024 0.942 80 82.34 ± 0.16 41.14 ± 0.09 1.290 ± 0.016 0.980 85 82.37 ± 0.00 36.00 ± 0.02 1.596 ± 0.068 0.988 90 82.37 ± 0.00 33.49 ± 0.02 1.849 ± 0.083 0.988 95 82.38 ± 0.00 31.57 ± 0.03 2.052 ± 0.125 0.994 aAsymptotic standard error.

or reduce it to negligible levels. The effect of temperature on the tively. There was a general increase in the rate constant with in- inactivation rate constants was described using the Arrhenius creasing moisture content confirming the hypothesis that the heat equation and is shown in Figure 2. The inactivation rate constants stability of enzymes increases at low moisture contents. At higher increased with increasing temperature as expected. This temper- moisture contents, the increased availability of solvent water results ature dependence of the inactivation rate constant is explained by in an increased opportunity for denaturation of the enzyme. The the concept of activation energy (Morales-Blancas and others thermal stability of phytase at different initial moisture content

2002). The calculated activation energies, Ea in the temperature according to k values would therefore be as follows: 35% < 25% < range 70 to 95 °C were 33.3 ± 0.4, 37.9 ± 0.6, and 43.4 ± 0.3 kJ/mol, 10%. The inactivation energy, Ea was highly correlated with the respectively at 10%, 25%, and 35%. Peers (1953) reported an inac- moisture content (R2 = 0.972). This dependence on the moisture tivation energy value in the temperature range 55 to 65 °C of 171.5 content is also obvious from Figure 4 where increasing moisture kJ/mol for partially purified phytase in wheat. The typical range for content resulted in an increase in Ea. Because Ea decreases at the inactivation energy for enzymes is 209 to 628 kJ/mol (Parkin 1993). lower moisture contents, it suggests that the susceptibility of Enzyme activity relative to the unheated control was significant- phytase to thermal inactivation decreases at the lower moisture ly reduced by heating moistened cowpea flour. Increasing moisture content. Several results in literature have also shown this depen- content generally facilitated the thermal inactivation of phytase. dence of the activation energy for deteriorative reactions on the

The variation of the inactivation rate constant and inactivation en- moisture content of the particular food product (Labuza 1980; E: Food Engineering & Physical Properties ergy with moisture content are shown in Figures 3 and 4, respec- Buera and others 1984). The nonlinear curves obtained for the inactivation of phytase could have resulted from thermal inactivation being a water-catalyzed reaction dependent upon successive

Figure 2—Thermal dependence of rate constant for thermal Figure 3—Effect of moisture content on loss of phytase ac- inactivation of cowpea phytase tivity in heated cowpea flour

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Table 3—Predicted and measured relative phytase activity (%) in heated cowpea flour Moisture content (%, dry basis) 10 25 35 Temp. (°C) Time (min) Measured Predicted Measured Predicted Measured Predicted 70 5 93.22 92.98 76.75 76.76 65.66 67.10 15 89.06 91.44 71.73 73.94 62.41 62.37 95 5 73.27 74.97 47.18 50.26 38.29 38.95 15 69.33 70.27 46.80 46.14 38.28 38.42 R2: 0.995 0.994 0.999

sorption of water molecules onto the protein (Multon and Guilbot used to successfully describe and predict the residual phytase ac- 1975). The temperature sensitivity of enzyme inactivation is directly tivity in cowpea flour. related to the water content of the system because water is needed This study was carried out to provide information on the factors to facilitate the unfolding of the protein during denaturation. En- that significantly impact the inactivation of cowpea phytase and zymes in dry or semi-moist foods tend to be more heat stable (Par- further apply this knowledge in developing heat processes for cow- kin 1993). A similar effect of moisture content on enzyme inacti- pea seeds. Overall, the results indicated that the highest degree of vation has been reported in several studies on other enzymes. inactivation was achieved under conditions of high temperature, These include the inactivation of phospholipase in soy beans (List high moisture content, and within the 1st 4 min of exposure to heat. and others 1990), myrosinase in canola seeds (Owusu-Ansah and To strike a balance between achieving a sufficient degree of phytase Marianchuk 1991), and lipase in rapeseed (Ponne and others 1996). inactivation (to affect its contribution to cowpea hardening), and retaining as much of the physicochemical characteristics of the Experimental validation of results and predictions treated cowpeas; the intermediate moisture content (25%) used in

E: Food Engineering & Physical Properties To test the validity of the proposed model, determinations of this study would be selected for further study. Additionally, the phytase activity were carried out experimentally in cowpea flour cowpea seeds would be briefly exposed to heat treatment because heated at 2 temperatures (70 and 95 °C) for 5 and 15 min for all the the decrease in phytase activity occurs fairly quickly. moisture contents studied previously. The residual phytase activity in the sample was then determined and compared with the values Acknowledgments predicted by the model using the same factors. From the results This study was supported by a grant from the Bean/Cowpea Col- (Table 3), it is observed that there is a close agreement between laborative Research Support Program, U.S. Agency for Intl. De- predicted and experimental values, suggesting that the fractional velopment (Grant nr GDG-G-00-02-0012-00). We thank CLP Pack- conversion model used is both reliable and valid under the exper- aging Solutions Inc. (Fairfield, N.J., U.S.A.) for their kind donation imental conditions. of retort pouches and Mr. Glenn Farrell for his assistance in setting up the thermal inactivation experiments. Conclusions hytase from cowpea flour exhibits a high heat resistance be- References Ptween moisture contents of 10% and 35% (dry basis). The ther- Anthon GE, Barrett DM. 2002. Kinetic parameters for the thermal inactivation of quality-related enzymes in carrots and potatoes. J Agric Food Chem 50:4119–25. mal inactivation of the enzyme is highly dependent on the heating AOAC. 1980. Official Methods of Analysis, 13th ed. Washington, D.C.: Assn. of Offi- temperature and moisture content of the medium. Although the in- cial Analytical Chemists. activation did not follow typical 1st-order kinetics, a modified 1st- Buera MP, Pilosof AMR, Bartholomai GB. 1984. Kinetics of trypsin inhibitory activity loss in heated flour from bean, Phaseolus vulgaris. J Food Sci 49:124–6. order model based on the fractional conversion technique was Cosgrove DJ. 1966. The chemistry and biochemistry of inositol polyphosphates. Rev Pure Appl Chem 16:209-24. Dvorakova J. 1998. Phytase: sources, preparation and exploitation. Folia Microbiol 43:323–38. Eeckhout W, De Paepe M. 1994. Total phosphorus, phytate-phosphorus and phytase activity in plant feedstuffs. Anim Feed Sci Technol 47:19–29. Greiner R. 2002. Purification and characterization of three phytases from germinat- ed lupine seeds (Lupinus albus var. Amiga). J Agric Food Chem 50:6858–64. Greiner R, Egli I. 2003. Determination of the activity of acidic phytate-degrading enzymes in cereal seeds. J Agric Food Chem 51:847–50. Hamada JS. 1996. Isolation and identification of the multiple forms of soybean phytases. JAOCS 73:1143–51. Hincks MJ, Stanley DW. 1986. Multiple mechanisms of bean hardening. J Food Tech- nol 21:731–50. Jones PMB, Boulter D. 1983. The cause of reduced cooking rate in Phaseolus vulgaris following adverse storage conditions. J Food Sci 48:623-49. Jongbloed AW, Kemme PA. 1990. Effect of pelleting mixed feeds on phytase activity and the apparent absorbability of phosphorus and calcium in pigs. Anim Feed Sci Technol 28:233–42. Kon S, Sanshuck DW. 1981. Phytate content and its effect on cooking quality of beans. J Food Proc Preserv 5:169–78. Labuza TP. 1980. The effect of water activity on reaction kinetics of food deteriora- tion. Food Technol 34:36-41,59. List GR, Mounts TL, Lanser AC, Holloway RK. 1990. Effect of moisture, microwave heating and live steam treatment on phospholipase D activity in soy beans and soy flakes. JAOCS 67:867–71. Liu BL, Rafiq A, Tzeng YM, Rob A. 1998. The induction and characterization of phytase and beyond. Enzyme Microb Technol 22:415–24. Lolas GM, Markakis P. 1977. The phytase of navy beans (Phaseolus vulgaris). J Food Figure 4—Changes in inactivation energy of phytase activity Sci 42:1094–7, 1106. with moisture content in heated cowpea flour Longe OG. 1983. Varietal differences in chemical characteristics related to cooking

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