E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V.

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Ultrasonic Inactivation of Soybean Trypsin Inhibitors
H. H. Liang^^ R. D. Yang^ and K. C. Kwolc^ 'Department of Applied Biology & Chemical Technology, University, Hong Kong Hong Kong Polytechnic

''Department of Bioengineering, South China University of Technology, Guangzhou, China.

Abstract Soybean trypsin inhibitors (TI) in soymilk were treated by heat and ultrasound of 20 kHz. The influence of several factors (temperature, time of treatment, pH, ultrasonic power, soymilk concentration, and ionic strength) on inactivation of TI was investigated. The results of the experiment shown that temperature was the major factor to affect the inactivation of TI, and treatment time was the next important factor. Under the experimental conditions of temperature 80 °C, ultrasonic power of 150 w, and pH 7.0 for 5 min treatment, TI in soymilk sample could be inactivated by 73%. The retained 27% TI was difficult to inactivate. This residual component is mainly Bowman-Birk inhibitor (BBI) which is extremely stable to heat as well as ultrasound of 20 kHz.

1. INTRODUCTION In soymilk processing, elimination of enzymic off-flavor development and destruction of growth inhibitors in raw soymilk are important concerns. Growth depression, pancreatic hypertrophy, hyperplasia and adenoma in experimental animals have been partly or fully attributed to soybean trypsin inhibitors (TI) [1]. Proper heat treatment improves the nutritional value of soymilk by inactivation of TI and by increasing the digestibility of soy proteins [2]. Previous reports on inactivation of TI in soymilk were mostly based on heat treatments [3]. Although heat is generally used to inactivate soybean TI, such inactivation is often incomplete. Heat treatment at temperatures below 100°C have shown that the inhibitors are rather heat stable, and it takes a long time to reduce the TI activity (TIA) to a satisfactory level. The use of high temperatures to destroy TI may lead to the degradation of amino acids and vitamins, development of off-flavor, and other deteriorative reactions. One of the important applications of ultrasound in food processing is its direct involvement into the processing, often in the form of high-intensity [4]. A number of beneficial effects have been reported in a broad aspects of applications, such as promotion of hydrolysis rates [5-6], assistance on diffusion [7], and destruction of micro-organisms [8]. Recently, the applications of manothermosonication (MTS), a technique of applying heat and ultrasound simultaneously under pressure, on inactivation of food spoilage enzymes have drawn more and more attentions of researchers. Lopez et al [9] studied the inactivation effects of MTS on some enzymes and found that the enzyme destruction efficiency greatly increased with

622 ultrasonic wave amplitudes, and the static pressure did not seem to significantly affect the destructive effect of the process. Lopez and Burgos [10] further investigated the effects of sonication physical parameters, pH, KCl, sugers, glycerol and enzyme concentration on the inactivation of soybean lipoxygenase (LOX), an enzyme involved in off-flavor development in vegetable products, under MTS operation. Their resuhs suggest that MTS inactivates LOX by two different mechanisms, one associated with heat and the other with ultrasound. They also found that the effect of static pressure on the inactivation of LOX is not substantial. Our objective is to investigate the feasibility of applying ultrasound in combination with a mild heat treatment on the inactivation of TI in soymilk at atmospheric pressure and the effects of various influent factors such as temperature, duration of ultrasound treatment, ultrasound power level, pH value of the solution, soy solid concentration and ionic strength.

2. MATERIALS & METHODS 2.1. Preparation of soymilk Canadian no. 1 grade soybeans were soaked in deionized water (soybean-to-water 1:7) for 14 h at 5°C. The soaked beans, along with the soak water were blended in a Waring blender at high speed for 3 min . The slurries were diluted with deionized water and filtered through a nylon filter bag and the insoluble residue was discarded. The filtrate, designated as soymilk, was lyophilized in a laboratory freeze-drier for 48 h and the dried product was stored in a screw-cap test tube at 5°C for later use. 2.2. Sample preparation 0.75 g sample of freeze dried soymilk solids were dispersed in 50 ml of deionized water and stirred using a magnetic stirrer for 3 h. The reconstituted soymilk has a pH of 6.5 and a solid content of about 3%. The pH of the soymilk was adjusted to the desired value by adding 1 M NaOH or 1 M HCL for experiments of pH effect. 2.3. Heat treatment 25 ml of the soymilk sample reconstituted from freeze dried soymilk solids was filled into a screw-cap test tube and heated in a boiling water bath. When the temperature of soymilk reached the desired temperature, the tube was transferred to another water bath previously set at the desired temperature and held for a desired length of time (holding time). At the end of the holding time, the tube was immediately transferred to a cold water bath. 2.4. Heat and ultrasonic treatment For ultrasound treatment, the sample, after heating up to the desired temperature, was immediately transferred to a glass cylinder (35 ml volume), which was then placed into a cooling cell with cold water inlet and outlet. The flow rate of cold water could be adjusted to maintain the sample temperature within ± 1°C of the desired value during ultrasound treatment. The sound probe (with a 13 mm tip) of the ultrasonic processor (Acros Chimica, model CPX 600), along with a thermocouple, were inserted into the soymilk sample, about 1 cm from the bottom of the cylinder. The desired ultrasound power level was obtained by controlling the wave amplitude. The operation mode of the ultrasound processor was set as

623 pulse with the on-off ratio of 3:2 second. At the end of the treatment, the cylinder with soymilk was immediately immersed into ice water. The treated sample was then diluted and the residual TIA was assayed. 2.5. Assay for trypsin inhibitor activity (TIA) A modification of the Kakade's procedure, developed by Smith et al. [11], using the synthetic substrate benzoyl-DL-arginine-p-nitroanilide (BAPNA), was employed to measure TIA. The method involves extraction of the inhibitors from the sample at pH 9.5 and mixing unfiltered suspensions with bovine trypsin. The activity of the remaining trypsin is then measured by offering it BAPNA under standard conditions. The p-nitroaniline released is measured spectrophotometrically at 410 nm. This provides a linear measure of the residual trypsin activity, so that the amount of pure trypsin inhibited per unit weight of sample can be calculated.

3. RESULTS i& DISCUSSION 3.1. Effect of temperature on ultrasound inactivation of TI Figure 1 gives a comparison between the effect of heat treatment alone and the combined effect of heat and ultrasound treatment on the inactivation of TI in soymilk. The results showed that the inactivation of TI was facilitated when ultrasound treatment was simultaneously applied with heat. The synergistic effect was found to be the highest at about 70 °C. One possible mechanism is that sonification gives rise to H- and OH- free radicals by decomposition of water inside the oscillating bubbles [12]. Hydroxyl radicals are very reactive and can induce the initial formation of peroxy radicals on amino acid residues, producing great losses of tryptophan, tyrosine, and other amino acids [13]. The hydroxl radicals may also cleave the disulfide bonds of the TI [14]. Another possible mechanism is that the vapor pressure inside the cavitation bubbles increases as the treatment temperature increases. The high pressure and temperature cause intensive collapse of the cavitation bubbles, that consequently increase the reaction rate of sonochemistry [15]. However, when the temperature was too high (90 °C in Figure 1), early collapse may occur due to the excessive high vapor pressure inside the cavitation bubbles, resulting in a the smaller ultrasonic effect on the inactivation of TI. 3.2. Effect of ultrasound treatment time on inactivation of TI Figure 2 shows the effect of sonification time on the inactivation of TI when the other influent factors are set as constants. It was found that the ultrasound inactivation effect was rapidly raised when the treatment time was increased from time zero to 5 min. The percentage of residual TIA declined from 100% to 32% during this period. However, sonification time longer than 5 min. shows adding no extra effect on the inactivation of TI. Experiment conducted at temperature of 80 °C also shown the similar trends. It is well known that there are two types of trypsin inhibitor in raw soybean [16], namely Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). The content of KTI in soybean is about 1.4%) and that of BBI is about 0.6%. The KTI and BBI contain two and seven disulfide bonds respectively. Since disulfide bonds stabilize the native conformations of proteins [17], thus BBI is much more stable than KTI to the effect of varying conditions

624 such as heat, acids and alkaUs. The experimental results indicated that BBI is also extremely stable towards the effect of ultrasound. Therefore, under the operation of ultrasound, about 30% of residual TIA is always difficult to inactivate.







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Figure 1. Effect of temperature on TIA when treated by: • -heat treatment; ^ -heat & ultrasound with power 150 W for 8 min.

Figure 2. Effect of sonication time on TIA when treated with power 160 W and at: O - 70 °C; • - 80 °C

3.3. Effect of ultrasound power on the inactivation of TI The effect of ultrasound power on the inactivation of TI is shown in Figure 3. When the applied power is lower than 130 W, the inactivation effect of TI is readily increased as the ultrasound power increases. The effect of ultrasound on the inactivation of TI recedes as the power of 150 W or higher is applied. This may be explained as that ultrasound power of about 150 W is enough to inactivate most KTI, while BBI is not affected by ultrasound power up to 170 W at given experimental conditions. 3.4. Effect of pH on ultrasound inactivation of TI Soybean TI was found to be more heat labile under alkaline conditions [15]. Heating in alkaline solution may cause more rapid destruction of disulfide bonds, which are important in the stability of soybean TI. However this phenomenon was not observed when heat and ultrasound treatment were applied. Figure 4 shows that inactivation of TI in soymilk is more effective at neutral pH than at acidic or alkaline conditions. 3.5. Effect of cosolutes on ultrasonic inactivation of TI Previous studies have shown that the stability of proteins in aqueous solution may be affected by the presence of cosolutes [10, 18]. The effects of soymilk concentration and NaCl concentration on the inactivation of TI are shown in Figures 5 and 6 respectively. Figure 5 indicated that the effect of solid contents of soymilk on the inactivation of TI is less profound compared to other influent factors. Figure 6 shows that NaCl concentration up to 0.5 M

625 facilitated the ultrasonic inactivation of TI significantly. For soymilk, concentration of NaCl higher than 0.5 M will affect its taste and no further investigation was carried out at such high NaCl concentration.



30 60 90 120 150 180 U L T R A S O U N D P O W E R (W)

Figure 3 Effect of ultrasonic power on TIA when treated at 70 °C and pH 7.0 for 5 min.

Figure 4. Effect of pH on TIA when treated by heat & ultrasound at 70 °C & power 160 W for 5 min.


1 2






S O Y M I L K S O L I D C O N T E N T (%)

0.0 0.1 0.2 0.3 0.4 0.5 0.6 N a C l C O N C E N T R A T I O N (M)

Figure 5. Effect of soymilk concentration on TIA when treated by heat & ultrasound

Figure 6. Effect of ionic strength on TIA when treated by heat & ultrasound

626 4. CONCLUSIONS This study shows that the appHcation of ultrasound with mild heat treatment at atmospheric pressure can facilitate the inactivation of TI. Treatment temperature is the most important factor in such operation. Other influent factors are sonification time, ultrasound power, pH of the solution and ionic strength. Under the condition of temperature 80 °C, ultrasonic power 150 W, and pH 7.0, about 73% of TI was inactivated for 5 min treatment. About 27% residual of TI was difficult to inactivate. This residual component is mainly Bowman-Birk inhibitor (BBI) which is extremely stable to the ultrasound of 20 kHz.



This work was financially supported by the Research Committee of Hong Kong Polytechnic University.

6. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 I.E. Liener and M. Kakade, Toxic Constituents of Plant Foodstuffs, Academic press. New York, 1980. A.K. Smith and S.J. Circle (eds.), Soybeans: Chemistry and Technology, Vol. I, Proteins, AVI Publishing, Westport, CT, 1972. K.C. Kwok, W.H. Qin, and J.C. Tsang, J. Food Sci. 58 (1993) 859. D. J. McClements, Trends Food Sci. & Tech., 6(9) (1995) 293. S. Barton, C. Bullock and D. Weir, Enzyme & Microbial Tech., 18(3) (1996) 190. M. Sakakibara, D. Wang, R. Takahashi, and S. Mori, Enzyme & Microbial Tech., 18(6) (1996) 444. J. D. FlorosandH. Liang, Food Tech., 48(12) (1994) 79. H. S. LiUard, Food Tech., 48(12) (1994) 72. P. Lopez, F. J. Sala, J. L. de la Fuente, S. Condon, J. Rosa and J. Burgos, J Agri. & Food Chem. 42(2) (1994) 252. P. Lopez and J. Burgos, J. Agri. & Food Chem., 43(3) (1995) 620. C. Smith, W. Van Megen, L. Twaalfhoven, and C. Hitchcock, J. Sci. Food Agric. 31 (1980)341. E.A. El'piner, A.V. Sokolskaya, and M.A. MarguHs, Nature 208 (1965) 945. K. Davies, M. Delsignore, and S. Lin, J. Biol. Chem. 262 (1987) 9902. N.K.D. Kella and J.E. Kinsella, J. Biochem. Biophys. Methods 11 (1985) 251. T. J. Mason and J. P. Lorimer, Sonochemistry, EUiis Horwood Ltd., Chichester, England, 1988. M. Jr Laskowski and I. Kato, Annu. Rev. Biochem. 49 (1980) 593. H. Neurath and R. L. Hill (eds.). The Role of sulfur in proteins (3rd ed.). Academic Press, New York, 1977. S. Timasheff, Annu. Rev. Biophys. Biomol. Struct., (1993) 67.

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