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Effects of calcium and high pressure on soybean proteins A calorimetric study F Speroni a M C A na M de Lamballerieb aCIDCA CONICET Facultad de Ciencias Exactas UNLP Calles 47 y 116 La Plata Argentina bGEPEA UMR CNRS 6144 ONIRIS BP 82225 44322 Nantes cedex 3 France a r t i c l ei n f o Article history Received 22 December 2009 Accepted 23 March 2010 Keywords Soybean protein High pressure Calcium Calorimetry a b s t r a c t The effects of calcium and high pressure HP treatment on the thermal properties of soybean proteins were analyzed in soybean protein isolate SPI a b conglycinin enriched fraction 7SEF a glycinin enriched fraction 11SEF and whey protein concentrate WPC For b conglycinin the temperature of denaturation Td decreased with up to 12 5 mM or 6 2 mM calcium in SPI and 7SEF respectively This parameter increased when calcium was more concentrated The Td of glycinin increased for every assayed calcium concentration The values of changes in Td DTd depended on calcium concentration and the proportion of b conglycinin and glycinin in the samples Activation energy decreased for glycinin in the presence of calcium HP treatment promoted denaturation of b conglycinin and glycinin Calcium protected both proteins in SPI 7SEF and 11SEF at 200 MPa and protected glycinin in SPI and 7SEF at 400 and 600 MPa Nevertheless calcium increased the degree of denaturation of b conglycinin in 7SEF at 600 MPa In the absence of calcium partially HP denatured polypeptides exhibited the same or lower Td than controls whereas in its presence they exhibited higher Tds than their respective controls 2010 Elsevier Ltd All rights reserved 1 Introduction The use of soybean proteins in the formulation of foodstuff is growing in western countries due to their nutritional and func tional properties Moreover the consumption of these proteins or protein associated compounds e g isofl avones is linked with var ious health benefi ts Aqueous extractable soybean proteins may be classifi ed as storage proteins or whey proteins by separation occur ring at pH 4 5 4 8 Sorgentini Koshiyama Kikuchi Rackis Wolf Iwabuchi and Yamauchi 1987b suggest that this phenomenon is due to the formation of complexes with the 7S globulins In some cases soybean proteins are combined with calcium in order to modify their functional properties and increase the nutri tional value of the fi nal product Chaiwanon Puwastien Nititham yong Puppo et al 2004 but little is 0963 9969 see front matter 2010 Elsevier Ltd All rights reserved doi 10 1016 j foodres 2010 03 022 Corresponding author E mail address marie de lamballerie oniris nantes fr M de Lamballerie Food Research International 43 2010 1347 1355 Contents lists available at ScienceDirect Food Research International journal homepage known about the effects on this phenomenon of the composition of the medium in which the proteins are dispersed There is some evi dence concerning the effect of the presence of cosolvents for example calcium increased the survival of HP treated bacteria probably by stabilizing the plasmatic membrane Black Huppertz Fitzgerald 200 400 or 600 MPa in the absence or presence of calcium dispersions of SPI 7SEF 11SEF and WPC at pH 8 10 w w protein 80 mg of dry matter Samples were heated in the calorimeter from 20 to 112 C at 1 C min A second scan was carried out on samples to check dena turation reversibility Samples were hermetically sealed in hastel loy pans Distilled water was used as the reference Enthalpie changes of thermal denaturation DH were estimated as the area between the DSC curve and a straight line extended between the onset and the fi nal temperatures of transition The temperature of maximum heat absorption Td and the enthalpy change of each peak were determined by Micro DSC III software and expressed as J g of protein dry matter basis Peak Fit software V4 0 Jandel Scientifi c Software Chicago IL was utilized to deconvolute the curves in order to know the percentage ofDH corresponding to b conglycinin and glycinin respectively The results are the mean of three calorimetric measurements In the present work the degree of denaturation was calculated as 100 minus the quotient betweenDH measured after treatment DHmeasuredwithouttreatmentforanygivencalcium concentration 2 5 Kinetic parameters Kinetic parameters for denaturation were calculated by the dy namic Ozawa method Ozawa 1970 This assumes that denatur ation kinetics follow Arrhenius s expression and it is based on the relationship between the variation of the maximum peak defl ection and the heating rate according to the expression ln b Td2 AEa R Ea RTd where b is the scanning rate Td is the temperature of denaturation Eais the activation energy A is the pre exponential factor and R is the universal gas constant Thermal scans were carried out at 0 1 0 2 0 5 0 6 and 1 0 C min for SPI 7SEF and 11SEF in the absence or presence of calcium 1348F Speroni et al Food Research International 43 2010 1347 1355 2 6 Statistical analysis Each treatment was performed in at least triplicate The statis tical analysis was completed using the Sigmastat software Systat Software USA Analyses of variance were conducted Differences between the sample means were analyzed by Tukey s test at an alevel of 0 05 3 Results and discussion 3 1 Protein composition of SPI 7SEF 11SEF and WPC Soybean protein isolate SPI contained b conglycinin and gly cinin in similar proportions and small amounts of lectin and Kunitz trypsin inhibitor Fig 1 The SPI protein content determined by the Kjeldahl method was 86 6 w b conversion factor of 5 8 7SEF contained b conglycinin in a high proportion 75 and glycinin 25 and its protein content was 84 5 w b 11SEF was almost exclusively constituted by glycinin with a protein content of 93 0 w b The more abundant proteins in WPC were lectin and Ku nitz trypsin inhibitor and its protein content was 61 6 w b 3 2 Effects of calcium on the thermal properties of soybean proteins 3 2 1 Denaturation temperature 3 2 1 1 Denaturation temperature of b conglycinin and glycinin in different soybean protein preparations Two typical thermograms of SPI are shown in Fig 2 A control sample was heated at 1 C min from 20 to 112 C and two endothermic peaks were observed cor responding to the denaturation of b conglycinin and glycinin When 25 mM CaCl2was added the two peaks were shifted to high er temperatures and a small shoulder appeared at 65 C Fig 2 arrow The Tds of b conglycinin in SPI and 7SEF and those of glycinin in SPI 7SEF and 11SEF at different calcium concentrations are shown in Fig 3 In the absence of calcium the Td of b conglycinin was 71 3 0 1 C with no differences between SPI and 7SEF Neverthe less a dependence of the Td of glycinin on the composition of the product was observed its value was 84 1 0 1 C in 11SEF and 7SEF whereas it was 86 9 0 2 C in SPI This difference was also reported by Petruccelli and A n 1996 and by Renkema Lake mond de Jongh Gruppen and van Vliet 2000 and is probably due to polypeptide interactions that increase thermal stability of glycinin in SPI This phenomenon might be related to differences in the preparation processes of 11SEF 7SEF and SPI which would modify protein structure leading to differences in the Td of glycinin 3 2 1 2 Calcium b conglycinin interaction The effect of calcium was also analyzed for b conglycinin and a two phase behavior was ob served for the dependence of Td on calcium levels Fig 3 panel a Up to 12 5 mM in SPI or 6 25 mM in 7SEF calcium addition pro duced a decrease in Td while from these values up to 25 mM a pro gressive increase was observed These results are in agreement with those of Scilingo and A n 1996 who also reported a pro gressive increase in the Td of glycinin and a two phase behavior for b conglycinin as calcium concentration increased in SPI disper sions A destabilizing effect of calcium on storage proteins was also described for Vicia Faba by Arntfi eld et al 1986 at a very high calcium concentration 2 M No changes in Td were reported by these authors at lower concentrations In our case the decrease in Td of b conglycinin was observed in both SPI and 7SEF The min imum in the Td of b conglycinin appeared in 7SEF at a lower cal cium concentration than in SPI indicating that the effect of calcium depends not only on its concentration but also on the pro portions of b conglycinin and glycinin 97 66 45 30 20 14 A B AA B KTI KTI L B PSPI 7SEFWPCP11SEFPSPI 7SEFWPCP11SEF 97 66 45 30 20 14 A B AA B KTI KTI L B Fig 1 Electrophoretic profi les of soybean protein isolate SPI b conglycinin enriched fraction 7SEF glycinin enriched fraction 11SEF and whey protein concentrate WPC a0 aand b are polypeptides of b conglycinin A and B are polypeptides of glycinin L lectin KTI Kunitz trypsin inhibitor S standard proteins 60708090100 2 3 4 SPI 25 mM CaCl2 Heat Flow mW Temperature C SPI Fig 2 DSC thermograms of the SPI control and SPI with 25 mM CaCl2added Heating rate was 1 C min The arrow indicates a shoulder in presence of 25 mM CaCl2 F Speroni et al Food Research International 43 2010 1347 13551349 3 2 1 3 Calcium glycinin interaction The effect of calcium on gly cinin was analyzed for several ion concentrations Fig 3 panel b In the case of 11SEF CaCl2was increased up to 3 75 mM At higher concentrations instantaneous phase separation occurred due to precipitation thus preventing the correct DSC determina tions It is remarkable that in SPI and 7SEF where both glycinin and b conglycinin are present calcium concentration was in creased up to 25 mM without phase separation the dispersions remaining stable during the DSC assays In the cases of SPI and 7SEF a partition of calcium was established according to different association equilibria with glycinin or b conglycinin Glycinin exhibits a higher affi nity and a larger number of specifi c binding sites for the ion than does b conglycinin Appu Rao a part of these complexes was soluble and the other was insoluble but remained in suspension the dispersions became turbid but spontaneous phase separation did not occur in 48 h An increase in calcium concentration produced a stabiliza tion of glycinin increase in Td in SPI 7SEF and 11SEF Fig 3 panel b The peak corresponding to glycinin in 7SEF 12 5 mM CaCl2was wider than at any other calcium concentration pre venting a precise determination of Td It seems that a second tran sition appeared at a higher temperature than expected Fig 4 panel a arrow To verify this phenomenon the temperature scan was performed at 0 1 C min In these conditions the denatur ation peak of glycinin appeared split Fig 4 panel a The same heating rate was tested for SPI 18 7 mM CaCl2and a shoulder also appeared in the peak of glycinin Fig 4 panel b arrow These results suggest that either there is a different denaturing mecha nism at these glycinin Ca2 ratios or there are multiple species of glycinin Ca2 complexes with different Tds As expected when the scans were carried out at the lowest heating rate the transi tions took place at lower temperatures than at 1 C min Hendrix Griko and Privalov 1996 found that fora lactalbumin at low protein and calcium concentrations two discrete species called apo free and holo calcium bound a lactalbumin were present and exhibited different Tds These data suggest that when there are specifi c interactions different Ca2 protein species may co exist at certain calcium protein ratios In our case a specifi c inter action between the imidazole group of the histidines from glycinin and Ca2 has been described Appu Rao Sorgentini Wagner 1999 and this temperature was not altered by the presence of 25 mM CaCl2 Fig 5 Lectin and Kunitz trypsin inhibitor remained in SPI in small proportions in accordance with the results reported by Sorgentini and Wagner 1999 Taking into account these data we concluded that the shoulder observed at 65 5 C in the SPI 25 mM CaCl2thermograms Fig 2 corresponds to KTI The denaturation peak of KTI was detected in these conditions because the peak of b conglycinin was shifted to a higher temperature while that corresponding to KTI did not change signifi cantly Sor gentini and Wagner 1999 also observed that the Td of KTI was very little altered by changes in NaCl concentration between 0 and 1 M 3 2 2 Kinetic parameters of the denaturation of b conglycinin and glycinin Ozawa s model was applied to the experimental data and the results showed a good fi t of the experimental values with the mod el since r values ranged between 0 962 and 0 999 These data sug gest that soybean protein denaturation is kinetically determined The results show that the Ea of glycinin is higher than that of b conglycinin when it is expressed as J mol of protein The differ ence in Ea values between glycinin and b conglycinin decreased when the values were expressed as J g of protein suggesting that the molecular mass and probably the number of interactions play an important role Table 1 In the case of glycinin Ea depended on the protein composition of the samples the highest value being found in SPI without added calcium The presence of calcium de creased Ea by approximately 25 in SPI 7SEF and 11SEF On the other hand Ea values of b conglycinin were not affected by cal cium in SPI or in 7SEF This result is in accordance with Scilingo and A n 1996 who analyzed the Ea of b conglycinin in SPI Ea values of b conglycinin were similar in SPI and 7SEF suggesting that this parameter is not dependent on the proportion of the two proteins The difference in sensitivity to calcium of glycinin and b conglycinin may be due to differences in the affi nity for the ion 3 2 3 Denaturation enthalpy of soybean proteins The enthalpy changes DH corresponding to the denaturation of total proteins present in the protein preparations are shown in Fig 6 The increase in calcium concentration was accompanied by an increase inD H This effect was signifi cant for SPI only at 25 mM calcium p 0 05 and for 7SEF from 12 5 mM p 0 05 This result suggests that calcium reinforces the protein structure by increasing the stability of existing interactions and or promot ing the formation of new ones like hydrogen bonds electrostatic interactions and or calcium bridges 304050607080 0 5 0 0 0 5 25 mM CaCl2400 MPa 0 CaCl2400 MPa 25 mM CaCl20 1 MPa Heat Flow mW Temperature C 0 CaCl20 1 MPa Fig 5 DSC thermograms of whey protein concentrate in the absence or presence of 25 mM CaCl2 for control 0 1 MPa or high pressure treated 400 MPa samples Table 1 Activation energy Ea for soybean proteins at different CaCl2concentrations in SPI 7SEF and 11SEF Values are expressed as average standard error ProteinSampleCaCl2 mM Ea J mol Ea J g b conglycininSPI0470368 9 6052 62 61 0 03 25449898 7 3271 12 50 0 02 7SEF0458576 0 24806 22 55 0 14 12 5432734 9 20792 32 40 0 12 25438685 5 11322 42 44 0 06 GlycininSPI01133382 6 72793 33 15 0 20 25918174 4 26091 32 55 0 07 7SEF0975061 8 51199 92 71 0 14 25722038 5 43658 42 01 0 12 11SEF0994595 8 59307 42 76 0 16 3 75726524 6 65040 92 02 0 18 0510152025 10 11 12 13 14 15 16 17 18 19 SPI 7SEF 11SEF H J g CaCl2 mM Fig 6 Enthalpy of denaturation DH of total proteins present in soybean protein isolate SPI b conglycinin enriched fraction 7SEF and glycinin enriched fraction 11SEF as a function of CaCl2concentration F Speroni et al Food Research International 43 2010 1347 13551351 3 3 Effects of HP and calcium on the thermal properties of soybean proteins 3 3 1 Denaturation The thermograms of non treated and HP processed in the ab sence or presence of calcium dispersions of SPI 7SEF and 11SEF are shown in Fig 7 Depending on the intensity of the HP the endo thermic peaks exhibited smaller areas compared with the controls at the same calcium concentration indicating denaturation Dena turation of soybean proteins by HP was also described by Puppo et al 2004 and Molina et al 2001 After treatment at 400 MPa in the absence of calcium the peak corresponding to glycinin almost disappeared in SPI and com pletely disappeared in 7SEF On the other hand b conglycinin con tained a signifi cant percentage of HP resistant structure nearly 30 after 600 MPa in SPI and 7SEF These results are in accordance 1 2 3 4 600 MPa 400 MPa 200 MPa Heat Flow mW Temperature C 0 1 MPa a 2 3 4 5 6 600 MPa Heat Flow mW Temperature C 400 MPa 200 MPa 0 1 MPa e 1 2 3 4 Heat Flow mW Temperature C 600 MPa 400 MPa 200 MPa 0 1 MPa c 5 4 3 2 1 0 1 MPa 200 MPa 600 MPa 400 MPa Heat Flow mW Temperature C f 1 0 1 2 Heat Flow mW Temperature C 600 MPa 400 MPa 200 MPa 0 1 MPa b 405060708090100110 405060708090100110 405060708090100110 405060708090100110 405060708090100110 405060708090100110 7 8 9 10 600 MPa 400 MPa 200 MPa Heat Flow mW Temperature C 0 1 MPa d SPI SPI CaCl225 mM 7SEF 7SEF CaCl225 mM 11SEF 11SEF CaCl23 7 mM Fig 7 DSC thermograms of soybean protein isolate SPI a and b b conglycinin enriched fraction 7SEF c and d and glycinin enriched fraction 11SEF e and f in the absence or presence of CaCl2 after different high pressure treatments Heating rates were 1 C min 1352F Speroni et al Food Research International 43 2010 1347 1355 with those of Puppo et al 2004 and Wang et al 2008 indicating that glycinin is more sensitive to HP than b conglycinin and refl ecting differences in the structure aggregation and dissociation phenomena that occur in these proteins during HP It is noticeable that when SPI was treated at 400 and 600 MPa in the presence of 25 mM CaCl2 the peak corresponding to KTI was more evident than in the non HP processed samples Fig 7 panel b arrows This result indicates that the peak of KTI is more marked when the peak of b conglycinin is reduced by partial denaturation and temperature shifted by the presence of calcium It also sug gests that the protein structure of KTI is HP resistant To confi rm this hypothesis samples of WPC were thermally scanned without or after a 400 MPa treatment in the absence and presence of 25 mM calcium It was observed Fig 5 that neither the Td nor DH of this peak was affected by HP Thes