Jw. | Food Science and Technology international (1998) 4, 79-84 Influence of enzymes, pH and temperature on the kinetics of whey protein hydrolysis Influencia de los enzimas, pH y temperatura en la cinética de la hidrélisis de las proteinas del lactosuero F. Camacho, P. Gonzélez-Tello and E. M. Guadix’ Departamento de Ingenieria Quimica Facultad de Ciencias Universidad de Granada 18071 Granada, Spain The influence of pH, temperature and the mixture of enzymes (MKC Protease 660 L and PEM 2500 $) on the enzymatic hydrolysis of whey proteins was studied. The experiments show that all results were reproducible via a kinetic model that supposes the rapid and irreversible binding Of part of the proteases to an inhibitor in the substrate, followed by a zero-order hydrolysis with respect to the substrate which occurs simultaneously with a second order enzymatic denatural- ization produced by an attack of the free proteases upon those bound to the substrate-enzyme ‘complex, Use of the optimum operating temperature of 60°C and pH 8-10 led to a greater degree | of hydrolysis. However, increasing the pH to these levels means that the salt content, on neutral- ining the hydrolysate, is somewhat high and this is often unsuitable for the preparation of special diets. In the experiments performed with mixtures of enzymes, two contrasting phenomena ‘occurred; there appears to be synergism between the proteases, which is preceded by a loss in enzymatic activity greater than that which can be accounted for by the presence of the inhibitor in the whey proteins. | Keywords: whey, proteins, hydrolysis, kinetics, enzymes Se ha estudiado la influencia del pH, temperatura y mezcla de enzimas (MKC Protease 660 Ly PEM 2500 $) sobre la hidrlisis enzimatica de proteinas del lactosuero. Los ensayos realizados pusieron de manifiesto que en todos los casos, los resultados experimentales pueden reproducirse mediante un modelo cinético que supone la unién répida e irreversible de una parte de las proteasas con un inhibidor presente en el sustrato, seguida de una hidrélisis de orden cero respecto al sustrato, simulténea con una desnaturalizacién enzimética de segundo orden provocada por el ataque de las proteasas libres a las proteasas ligadas al complejo enzima sustrato. Se comprobs que la temperatura dptima de operacién era 60°C y que un aumento de pH, entre pHi=8 y 10, conducia a un mayor grado de hidrélisis. Sin embargo, el aumento de pH provoca un aumento del contenido en sales, después de neutralizar el hidrolizado, lo que resulta ser un inconveniente ‘cuando se quieren utilizar para la preparacién de dietas especiales. En los experimentos realizados ‘con mezclas de enzimas se dieron dos fendmenos contrapuestos, ya que parece existir un siner- _gismo entre las proteasas, que viene precedido por una pérdida inicial de actividad enzimatica mayor que la atribuible a la presencia del inhibidor en las proteinas séricas Palabras clave: suero, proteinas, hidrdlisis, cinética, enzimas INTRODUCTION temperature conditions and the hydrolysates obtained can be used in the preparation of baby food and special diets for enteric nutrition (ost et a, 1989). The hydrolysates produced in this way must be hypoal- lergenic, osmotically well balanced and be acceptable in taste (Otani et al, 1990; Rouanet et al, 1990). These ‘The enzymatic hydrolysis of whey proteins should be a mild process carried out under moderate pH and * To whom correspondence should be Sen | Reet 5 Perl 190, weed 1 fe 1997 properties depend’ on the greater fraction of the | —Ee——E—_———E—ereeeeeem LL _—hydrlysates being made up of peptides with a mole- 1082-0132 © 1998 Chapman & Hall —_— _—_ _—_———80 cular weight of between 500 and 1000 Da (Grimble et al,, 1986; De Freitas et al, 1993). The enzymatic hydrolysis of whey proteins using endoproteases leads to the production of peptides of varying sizes, which in turn form the substrate for further hydrolysis; for this reason it is difficult to follow the kinetics of each of the different substances formed (Postolache and Oncescu, 1989). Nevertheless, from a practical point of view, it is of greater interest to analyse the overall rate of hydrolysis in terms of the degree of hydrolysis achieved and of the operating conditions: initial enzyme and substrate concentrations, pH and temperature. With this overall kinetic equation it is possible to design the enzyme Feactor reasonably well. Furthermore, our experi- menial results show that it is possible to relate the degree of hydrolysis achieved with the distribution of molecular sizes in the hydrolysates (Gonzélez-Tello et al, 1994b). Thus, once the conversion in certain specific conditions has been determined it is possible to obtain the overall rate of hydrolysis and the distri bution of molecular size in the hydrolysates, all of Which information is of practical interest. In a previous study, Gonzélez-Tello et al. (1994a), studied the influence of the initial concentrations of substrate [S, (g/L)] and of enzyme [E, (AU/L)] at pH8 and a temperature of 50°C on the kinetics of the hydrolytic reactions for the whey proteins with proteases from different origins, bacterial and animal. In this work we have studied the influence of pH, temperature and the mixture of enzymes on this enzymatic hydrolysis to obtain the kinetic para- meters and the optimum operating conditions of the process. MATERIALS AND METHODS The substrate was a commercial preparation of whey proteins, Lactoalbumin 75 L (MILEI, Germany) containing a total of 73% N. The enzymes were MKC- Protease 660 L (Miles, Solvay Enzymes GmbH & Co. KG, Hannover) of bacterial origin, and PEM 2500 S (Novo Industries, Bagsvaerd, Denmark), of animal origin. The activity of these proteins was measured according to the Anson’s modified method (1978), and proved to be 6.6 AU/ml for MKC Protease 660 L and 20.84 AU/g for the PEM 2500 S. The characteristics of both the substrate and the enzymes have been described more fully in a previous paper (Camacho et al, 1992), ‘The kinetics of the enzymatic hydrolysis was followed experimentally in a stirred, tank-type, batch reactor and the degree of hydrolysis achieved (x) was F. Camacho et al determined via the pH-stat method (Adier-Nissen, 1985). The degree of hydrolysis is defined by the ratio between the number of hydrolysed peptide bonds and the total number of peptide bonds in the untreated protein; this can be measured by the equation: E Meh, where E, is the equivalent of 2 NaOH consumed to keep the pH constant, M, is the mass of protein (kg), +, i the total number of equivalents of peptide bonds by unit of protein mass. (h,= 8.8 meq/g for whey proteins: Novo Industries, 1989), and a is the degree of dissociation according to pH and temperature (Novo Industries, 1989). Gonzélez-Tello et al, (1994a) found that with either ‘endoprotease (MKC Protease 660 L or PEM 2500 5) the overall rate of hydrolysis, r, = Spx dx/dt, for the whey proteins can be explained by a kinetic model which includes the following hypothesis: (i) a rapid and irreversible binding of the active enzyme with the inhibitor present in the substrate. (The existence of a protease inhibitor in whey proteins was shown by Weber and Nielsen in 1991). ; w E+ ft @ where I is the concentration of inhibitor, EI is the concentration of the enzyme-inhibitor complex and k, is the inhibitory Kinetic constant (M1 §"), This determines that the initial concentration of the enzyme is: Ex = Ey-€° Sp ) where Ep, is the initial active enzyme concentration and ¢ is fraction of enzyme activity loss (AU/g protein); and (ii) zero-order hydrolysis with respect to the substrate, simultaneous with a second-order enzymatic denaturalization brought about by free enzyme attacking the enzyme bound to the substrate: E+S—+ES a ky ES—+ E+ 2P o E+ ESSE + Ey +S © where ES is the concentration of the enzyme-substrate complex, K, is the kinetic constant of hydrolysis (g/AU.min), K, is the kinetic denaturation constant (g/AU.min), P is the concentration of product and E is the concentration of deactivated enzyme. This mechanism leads to the following kinetic equation: FOOD SCIENCE AND TECHNOLOGY INTERNATIONAL (1998) 4(2)Hydrolysis of whey proteins a eg Lk) oa Soe = fy Eo~ € Sy oxp|- ne) where 1, is the overall rate of protein hydrolysis (g/L. min). RESULTS AND DISCUSSION Enzyme Mixtures The results obtained at pH 8 and 50°C with different mixtures of MCK Protease 660 L and PEM 2500 S are shown in Figures 1 and 2, It can be seen that the conversion (2) always increases concomitantly with the time of hydrolysis and that the overall hydrolysis rate decreases considerably the longer the process continues. This decrease in the overall hydrolysis rate may be caused by a scarcity of peptide bonds capable of being hydrolysed, by enzymatic inhibition, or by denaturalization of the proteases. The first factor has scant importance and it would seem that the latter two are those which control the kinetics of the hydrol- ysis (Guadix, 1993), as it has been shown that: mek E ®) and that the instantaneous concentration of active enzyme (E) diminishes exponentially during the 03: oz oa ° st 00280 Time (nin) Figure 1. Hydrolysis of whey proteins by mixtures of MKC-Protease 660 L and PEM 2500 S, pH = 8.0, Tem- perature = 50°C. (€} E,{P.660L) =33.0AUML, E,!P.2500 S$) =23.0 AUML, S,= 38.45 gil. (0) E,{P.660L) = 99.0 AUML, E,{P.2500 S) » 31.26 AUML, Sy = 109.5 g/L. (A) Ee(P-660L) = 3.30 AU/L, E\(P.2500 S)~ 16.12 UL, S, = 38.45 g/t. {-) Equation (9). Figura 1. Hidrdlisis de las proteinas del lactosuero con mezclas de MKC-Protease 660 L y PEM 2500 S, pH= 8.0, ‘Temperature = 50°C. (@) E,{P.660L) = 33.0 UAL, Ey(P.2500 S) = 24.18 UALL, S, = 38.45 g/L. (0) E,{P.660L) = 99.0 VAIL, E,(P.2500 S} = 31.26 UAL, S, = 109.5 g/L. (A) E,(P.660L) =330UA/L, EJP.2500 S)=16.12UAML, $,=38.45 g/l. (-) Eeuacién (8), 81 ° 3 = 8 wo 128 Time (in) Figure 2. Hydrolysis of whey proteins combined with mixtures of MKC-Protease 660 L and PEM 2500 S, pH=8.0, T~ 50°C. (@) E:(P.660L) = 99.0 AUIL, E,{P.2500 8) = 15.6 AUIL, S.~ 109.5 g/. (0) &,(P.660L) - 49.5 AUIL, E,(P.2500 S)= 15.6 AL/L, S,= 109.5 g/L. (-) Equation (9). (+) Equation (12). Figure 2. Hidrélisis de las proteinas del lactosuero ‘con mercias de MKC-Protease 660 L_y PEM 2500 S, pH = 8.0, T=50°C. (@) E(\P.660L) = 99.0 UAL, Eq(P.2500 §)= 15.6 UAML, S;= 109.5 gL. (0) E,(P.660L) = 49.5 UAL, E\(P.2500 S) = 15.6 UAL, S,= 109.5 git. (-) Ecuacion 8. (=) Ecuacién 12, conversion. This fact may attributed either to inhibi- tion by products of the reaction or to denaturalization of the free enzyme (Gonzéler-Tello et al, 1994a). In our experiments this latter factor would appear to be the most important. By integrating Equation (7) we get: fu in[r +t, (E-¢) ‘| ° and by applying this equation to our results we are able to calculate parameters k/ky and ky(E9/So ~ ©) for the enzyme mixtures assayed via non-linear regression (Table 1). In the light of these results it can be deduced that k,/k, is more or less constant, so that by taking a mean value we have recalculated the corresponding k,(E)/S, ~©) values (Table 1). The representation of this term versus E9/Sp fits reason- ably well to a straight line: ole (Bo dosye nos 7500 from the slope and origin of which it is possible to calculate k, and ¢ respectively; once these values are obtained it is possible to calculate f, (Table 2), and with these values being incorporated into Equation (9) the experimental results were reproduced within 2 margin of # 5%. P= 0999 (10) FOOD SCIENCE AND TECHNOLOGY INTERNATIONAL (1998) 42), —82 Table 1. Parameters for Equation {9) for mixtures of MKC-Protease 660 L and PEM 2500 S, pH 8 and temper: ature 50°C. Tabla 1. Parémetros de la ecuacién (9) para mezclas de MKC-Protease 660 L y PEM 2500 S, pH 8 y T 50°C. Exean Eom Sp EySp bk (E/Sp~ x hy au AUR gil AU min? 49.5 156 10950 0595 0.0328 2.50 99.0 156 109.50 1.047 0.0334 6.00 99.0 31.26 10950 1.188 0.0322 6.98, 3.3 1812 3845 0.505 0.0340 1.69 33.0 230 3845 1.456 0.0364 «8.81 Table 2. Kinetic parameters for Equation (9) for mixtures of MKC:Protease 660 L and PEM 2500 S. Tabla 2. Parémetros cinéticos de la ecuacién (9) para mezclas de MKC-Protease 660 L y PEM 2500S. Enzyme —_ky [Q/AUxmin)! « (AU/g) k, {Q(AUxmin!) Protease 660 0.241 0.025, 7.95" PEM 2500S 0.171 0.028 8.36" Mixture 0.254 0.265 781 Data from Gonzéler-Tello et af. (1994a), The value of k, for a mixture of MKC Protease 660 L PEM 2500 S is greater than that of the enzymes when they act separately. Conversely, the ky value for the protease mixtures is lower (Fable 2). In both cases, the values of the kinetic constants were closer for Protease 660 L, which is the more efficient enzyme, defined in terms of hydrolysed bonds/AU. On the contrary the value of ¢, the enzymatic activity frac- tion lost during the initial moments (AU/g protein) was considerably greater than that corresponding to the individual action of each of the proteases able 2). The values for k, and k, of the mixtures of Protease 660 L and PEM 2500 S indicate that their joint pres- ence increases the overall hydrolysis rate and would appear to suggest synergistic activity. On the other and, the value of ¢, which is considerably higher than expected, seems to indicate that the initial, instantaneous and irreversible loss in activity may be caused by a combination of the presence of an inhibitor in the substrate and by a deactivation brought about by the action of each enzyme on the other. F. Camacho et al The concentration of the inhibitor in the substrate for each of the enzymes acting alone is 6, Sp and &» $, (AU/ In the extreme case of the inhibitor being simulta- neously selective for both enzymes the total concen- tration would be: 6 Sp & Sy (AU/LD Nevertheless: 48) + 65) < Sp This fact appears to indicate that the instantaneous and irreversible overall loss of activity in both enzymes is due to the presence of the inhibitor and in addition to other deactivating phenomena, among, which may be the deactivation of one enzyme by the other, ie Ey + Eq ~ deactivated form of enzyme Although the possibility exists that this process ‘might occur throughout the hydrolytic process, it must be more important when the proteases are in solution, and when the concentration of soluble peptides is low (quasi-heterogeneous hydrolysis phase). During these initial phases, the enzymes attack each other and also produce the shorter-chain whey peptides, which form a substrate for both proteases (homogeneous hydrolysis phase). At this time, due to enzymatic selectivity, the proteases should hydrolyse the peptide chains of the whey proteins in preference. To determine which of the two effects predominates (either synergism or an initial decrease in enzyme con- centration) we compared our actual results with those that might be expected if the hydrolytic activity of the ‘enzymes was additive, Furthermore, so that the results would be conservative as far as any possible syner- gism is concerned, we presumed that the instanta- neous, irreversible decrease in activity corresponded entirely to the PEM 2500 S, which is the least effective of the enzymes assayed. Under these conditions Equation (7) becomes: dx 15 8) = hy, (Ey) 0x So Ge = by Ea) ep an Fig Boy = & S) exp [- when on separating variables and integrating: t= i 12) ——T &\ | ke FOOD SCIENCE AND TECHNOLOGY INTERNATIONAL (1998) 42)nyarolvsis of whey proteins wuting numerical values into Equation nd integrating numerically we can obtain x Jes ior any time point, Some of our results are yp ts an example in Figure 2. In both cases it can see that there is an initial loss of activity during wrst moments, which is reflected in ¢, and that veer a conversion of 10-12% the synergistic effect wins to predominate, induced by an increase in ky 2 decrease in ky. The influence of PH ‘The degree of hydrolysis increases concomitantly with an increase in pH and reaction time (Figure 3). The parameters of Equation (9) were calculated by the non-linear regression of x versus the time of hydrolysis, accepting that the pH had no influence ‘on the fraction of the'inhibitor in the substrate ‘¢ = 0.025 AU/g). The values for ky, and k, (Table 3), introduced into Equation (9) reproduce the experi mental results with margins of error of less than 5%; k, appears to have a minimum value at a pH close to @f and the ky/ky ratio increases linearly with pH: ky All our data, therefore, indicate a positive effect of the increase in pH on the hydrolysis rate, as ky dimin- ishes more than k,. Nevertheless, it must be born in = ~ 0.0545 + 0.0106 pH? = 0.999 a3) 02 —— 7 | i i | : = vec ‘Time tmin) Influence of pH on the hydrolysis of whey : (0) pH 9; (A) £,=3.3AUML, Figure 3. proteins by MKC-Protease 660L. (@) pHi ‘pH 10; (~) Equation (9). Temperature 50° Sy= 38.45 gil lnfluencia del pH en {a hidrétisis de las rotease 660 L. (@) n (9), Temperatura Figura 3. proteinas del lactosuero con MK\ 1H 8; (O) pH9; (4) pH 10; (-) Ecuac 50° C,£)= 3.3 VAIL, Sp 38.45 g/t. 83 Table 3. Influence of pH. Values for the kinetic con- tants in Equation (9) £,(P.660 L)= 3.3 AU/L Sy = 38.45 afl, ¢= 0.025 AUIa. ‘Tabla 3. Infivencia del pH. Valores de las constantes cinéticas, Ec (9) Ex{P.660 L)=3.3 VAIL S,= 38.45 g/l «= 0.025 UANg pH, (g/AUxmini) ky (ghAUxmi klk 8 0.241 7.98 0.0303 8 0.183 449 0.0408 10 0218 423 0.0515, mind that the higher the pH the greater will be the sall concentration in the hydrolysate, which will have to be neutralized later and may result in products unsuitable for dietary and infant foods. The influence of temperature ‘The overall rate of hydrolysis and the final conver- sion both increase concomitantly with temperature. (Figure 4). The parameters of Equation (9) were calcu- lated by non-linear regression (Table 4), the kinetic ‘constants correspond to the experimental ones by a margin of less than + 5%. The values for k, and ky showed that both these kinetic constants increase with temperature. Therefore, the processes of enzymatic hydrolysis and enzyme denaturation are enhanced by i ° 0 100 150 200 Tire (rin) Figure 4. Influence of temperature on the hydrolysis of whey proteins by Protease 660L. (@) 50°C; (©) 60°C; (A) 70°C; (-} Equation (9). pH 8, E=3.3AUIL, 5, = 38.45 gil Figura 4. Influencia de la temperatura en la hidrdlisis de las proteinas del lactosuero con Protease 660. (@) 50°C; (©) 60°C: (A) 70°C; (+) Equation (9). pHs, F,=3.3UAlL, $238.45 g/L FOOD SCIENCE AND TECHNOLOGY INTERNATIONAL (1998) 42)aa an increase in temperature. Thus the size of the ratio ky /k, expresses the overall influence of this factor on the process. Ay/ky Seems to be at its maximum at around 60 *C (Figure 5), which would therefore be the optimum temperature for operating the system Table 4, Influence of temperature. Valves for the kinetic constants in Equation (9) E, (P.660 L) = 3.3 AUML Sp = 38.45 gil «= 0.025 AU/g Tabla 4. Influencia de la temperature. Valores de tas consiantes cinéticas, Ec. (9) &, (P.660 L) =3.3UAL S,= 38.45 gf. « = 0.025 UA’g TC ky IGMAUE i ky [g(AU{min)| Kf 50 0.241 795 0.0303 60 o.a7e 1212 0.0394 70 028 25.94 0.0380 0.085 i i 0.04 | Zooed | £0085 | I i i o.02! 0.09 40 50 60. 70 80 “Temperature (°C) Figure 5. The variation of kyk, versus temperature, whey proteins-Protease 660 L system sistema proteinas del lactosuero-Protease 660 L F Camacho ot a, REFERENCES ‘Adler-Nissen J. (1985) Ensymie hydrolysis o fd protein. 12-19. London: Elsevier Applied Science Publishers Lo Anson A. 11978) Madied asonchenogobin methed for ay determination of protoltc actin. Nevo mndustias AE af ce. Camacho F.. 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(1991) Isolation and partial char- acterization of a native serinetype protease inkibitor from bovine milk, journal of Dairy Science 74: 764-771 FOOD SCIENCE AND TECHNOLOGY INTERNATIONAL (1938) 4(2) 4