Influence of Maltodextrins with Different Dextrose Equivalent on the

Chapter 21

Influence of Maltodextrins with Different Dextrose Equivalent on the Interaction between Hexyl Acetate and Legumin in an Aqueous Medium Maria G. Semenova, Anna S. Antipova, LarisaE.Belyakova, Yurii N. Polikarpov, Tamara A. Misharina, Margarita B. Terenina, and Rimma V. Golovnya

Downloaded by PURDUE UNIV on November 30, 2016 | http://pubs.acs.org Publication Date: September 7, 2000 | doi: 10.1021/bk-2000-0763.ch021

Institute of Biochemical Physics of Russian Academy of Sciences, Vavilov str. 28, 117813 Moscow, Russia

The effect of maltodextrins with different dextrose equivalent (Paselli SA-2 and SA-6) on the binding behavior of legumin relative to hexyl acetate (HxAc) has been studied by combination of ultrafiltration and gas-liquid chromatography. Both simple 1: 1 mixtures of legumin and maltodextrin as well as their heat-induced conjugates have been studied. A n analysis of the binding isotherms has shown that maltodextrin with the lowest dextrose equivalent (DE 2) has a rather high binding capacity in respect to HxAc and could be a real competitor with the protein in the binding of the aroma compound. In addition, the mechanism of HxAc binding with maltodextrins is distinctly different from that of legumin. A dramatic mutual effect of maltodextrins and legumin on binding behavior (the binding isotherms, the binding mechanisms, the intrinsic binding constants, and the number of binding sites) relative to HxAc has been found. The thermodynamic basis of the effect is discussed.

Most food systems contain both proteins and polysaccharides and the nature of their interactions is of fundamental importance to their functional properties in such systems (1 - 4). One of the principal functional properties of food proteins and polysaccharides is their capacity to bind aroma compounds. Proteins, as a rule, bind aroma compounds through hydrophobic interactions, whereas polysaccharides form inclusion complexes with aroma compounds much as starch does, for example (5-8). A better understanding of the key features of the mutual effect of protein and polysaccharide on their binding capacities for an aroma compound under a variety of experimental conditions, especially when the protein and polysaccharide were alone or in a complexed form, is of great importance.

260

© 2000 American Chemical Society

Roberts and Taylor; Flavor Release ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

261

Until recently, the mutual effect of polysaccharides and proteins on their capacities for binding flavor in an aqueous medium was not well understood and the prime object of this study has been to elucidate this effect. Commercially important polysaccharides (maltodextrins with different dextrose equivalent (DE)), legumin ( U S globulin of broad beans) and hexyl acetate (HxAc), being the major component of several attractive flavors, were selected as the main objects of our investigation.

Experimental


Materials Two commercial samples of maltodextrins (Paselli SA-2 and SA-6 , Avebe (the Netherlands)) were used as supplied. They are enzymatic products of the hydrolysis of a potato starch having D E values of 2 and 6, respectively. Legumin (1 IS globulin) was isolated from broad beans (var. "Agat") (7) and homogeneity of the isolated 1 IS globulin was assessed by sedimentation. A single peak of 1 IS globulin with a sedimentation coefficient of 12 χ 10" S was found. Hexyl acetate (HxAc) was purchased from R E A C H E M (Russia) (95% purity) and distilled to reach a purity of 99%. Organic solvents (> 99% purity), namely, acetone, ethanol, diethyl ether, were obtained from R E A C H E M (Russia) and used without further purification. Phosphate (pH 7.2) buffer solutions were prepared using analytical grade reagents (99.9 % purity) and double-distilled water. 13

Methods Determination of Hexyl Acetate Concentration in Solutions Concentration of aroma compound in solutions was determined using a Hewlett Packard 5710A gas-liquid chromatograph (integrator 3380A). Separation of extracted components was carried out using a fused silica capillary column SE-30 (JIW) (50 m χ 0.32 mm, df = 0.2 pm and/or 60 m χ 0.32 mm, df = 0.25 pm) at 110 °C. The operating conditions were as follows: a split ratio of the carrier gas (helium) was 1:30; rate was 1 mL/min, temperature of the injector and detector (FID) was 150 °C, injected samples ranged between 0.5 and 2 pL. The content of HxAc was calculated based on the relation between the peak areas of HxAc and an internal standard (nundecane). Preparation of Maltodextrin and Mixed Legumin-Maltodextrin Solutions Maltodextrins with different D E values were dissolved in an aqueous phosphate buffer (pH 7.2, ionic strength 0.05 mol/dm) by mechanical stirring at 85 °C for 1 hour. Thereafter the solutions were allowed to cool at room temperature. These solutions were used in all experiments and in the preparation of mixed solutions with legumin. The values of average molecular weight of maltodextrins, determined by light scattering in these solutions, were equal to 250 kDa and 102 kDa for SA-2 and SA-6, respectively (9). Concentration of maltodextrins and legumin in the solutions


262 were determined by evaporation at 105 °C to a constant weight. The protein or maltodextrin content in the solutions was assessed as the difference in the weight of the dry residues between a buffered solution containing biopolymer and the blank one.


Preparation of the Conjugate of Legumin with Maltodextrin Protein-polysaccharide conjugates were prepared as described in Ref. (10). The protein-polysaccharide mixed solutions were prepared as noted above and then freeze-dried. The freeze-dried legumin - maltodextrin mixtures were kept at 60 °C in the presence of a silica gel (45% relative humidity) for a period of 3 weeks. Preparation of Conjugate Solutions Samples of conjugates were dissolved in an aqueous phosphate buffer (pH 7.2, ionic strength 0.05 mol/dm) by mechanical stirring at 50 °C for 1 hour. Thereafter the solutions were allowed to cool at room temperature. Conjugate concentration was determined by evaporation at 105 °C to a constant weight. The conjugate content in solution was estimated as the difference in the weight of the dry residues between a buffered solution containing conjugate and the blank one. Polysaccharide concentrations in the conjugates were measured by the phenol-sulfuric method (77). Protein concentrations in the conjugates were estimated as the difference between the total concentrations determined by drying and the polysaccharide concentrations determined by the phenol-sulfuric method. Determination of HxAc Binding to Maltodextrins in Single Protein + Maltodextrin Mixtures and in the Protein + Maltodextrin Conjugates Hexyl acetate was chosen as an aroma ligand for the investigation. To determine the concentration of H x A c bound to the various samples, the ultrafiltration method was used to separate HxAc bound to the macromolecules from the free ligand. For binding experiments, concentrations of HxAc in solutions were varied from 0.5 to 2.75 m M . The calculated quantities of the aroma compound (in diethyl ether) were added into 25 m L of either maltodextrin solutions or the simple mixed solutions of maltodextrins with legumin or into the conjugate solutions as well as the same volume of an aqueous buffer being the blank solution. Concentration of the protein in the simple mixed solutions with maltodextrins was kept constant and equal to 1 % w/w. The concentration of the maltodextrins both in the binary (maltodextrin solvent) and in the simple mixed solutions with the protein was kept constant and equal to 1 % w/w. Concentration of each biopolymer in the conjugate solutions was determined as described above. Solutions with added HxAc were shaken for 1-2 minutes and left to stand for 30 minutes at room temperature in open flasks to allow ether to evaporate and then the closed flasks were shaken ( C P L A N water bath shaker, type 357, Poland) for 1 hour to reach equilibrium. The solutions were filtered through nuclear membranes with a pore size of 0.027 pm (the membranes were made from lavsan, Russian equivalent of Darcon, and were etched with 20 wt% NaOH. There was one carboxyl group for every 200 A of the membrane surface and the number of pores per cm was 2 χ 10 .) using a special equipment operated at 3 bar to separate free ligands from bound 2

2


9

263


ligands. Neither the protein nor the maltodextrins passed through the membrane. Aliquots of the filtered aqueous solutions (5 mL) were collected directly into test tubes containing 3 m L of diethyl ether. The two-layer mixtures were shaken and stored for 24 hours in a cool place. The organic layers were separated and used for determination of free ligand concentration in the filtrates by gas-liquid chromatography (GC). Sorption of HxAc by the biopolymers was estimated by G C from 3 - 8 experiments at each studied concentration of HxAc. About 20 experiments were carried out to estimate the sorption of HxAc by the nuclear membranes. The sorption of the aroma compound on the nuclear membrane was built into calculations to estimate the degree of bound ligand. The experimental error of a determination of the values of the aroma compound bound with macromolecules was 5 %.

Results and Discussion Before giving an account of the effect of maltodextrins on the binding capacity of the native legumin relative to HxAc in their simple mixtures or conjugates, let us elucidate the binding ability of the maltodextrins in an aqueous medium.

Binding of Hexyl Acetate with Maltodextrins with Different Dextrose Equivalents. Comparison with Binding of Hexyl Acetate with Legumin Figure l a shows binding isotherms of HxAc with maltodextrins with different D E values. The binding isotherms indicate considerably more binding (v is the number of moles of an aroma compound bound per mole of biopolymer) of H x A c by maltodextrin SA-2 than maltodextrin SA-6. This result agrees well with those obtained previously (72) and suggests an increase both in the number of binding sites and the affinity constant with increasing degree of polymerization of dextrins. It is also interesting to compare the binding isotherms of maltodextrins and legumin. Figure l a shows this comparison. Contrary to expectations, a great binding extent ν was found for maltodextrin SA-2 as opposed to legumin at rather high concentrations of H x A c in the system. As this takes place, maltodextrin SA-6 exhibits the least binding ability. B y this means maltodextrin SA-2 can play the role of a real competitor with the protein in binding of HxAc in mixed aqueous solutions. In addition, the binding data were presented in the form of a Scatchard's plot (75) in accordance with the following equation (14):

= nK-vK.

Here ν is the number of moles of ligand bound per mole of bipolymer, [L ] is the free ligand concentration in moles per liter; η is the total number of binding sites in macromolecule; Κ is the intrinsic binding constant (Figure lb). The specific shape of the curve (the magnitude of ν / [L ] grows with increasing ν in the range of low values of v) suggests the cooperative binding mechanism on the interacting binding sites for H x A c binding with both maltodextrin SA-2 and maltodextrin SA-6. This is more pronounced in the case of maltodextrin SA-2. This result is in reasonably good free

free


264


agreement with the experimental data found by Rutschmann and Solms (75), which pointed to a positive cooperative effect in the formation of the complex between starch and menthone, decanal or naphthol. Moreover, the Scatchard plots clearly show the difference in the mechanism of HxAc binding to maltodextrins, compared to legumin, where binding occurred on the independent and identical binding sites on the protein globule (14).

Figure 1. Binding of hexyl acetate with biopolymers in an aqueous medium (pH 7.2, ionic strength 0.05 mol/dm ): · , native legumin; •, maltodextrin SA-2; Ύ, maltodextrin SA-6. a) Binding isotherms: binding extent ν is plotted as a function of the logarithm of free ligand concentration in the system, log [Lf ]. b) Scatchard plot: the ratio of binding extent to free ligand concentration, ν / [Lf ], is plotted against binding extent v. 3

ree

ree

(See more details on the HxAc binding with legumin in the relevant chapter of this book). Unfortunately, an estimation of the binding parameters by the proper extrapolation of ν / [L ] to zero was impossible in the case of the binding according to the cooperative mechanism, because of the insolubility of H x A c in an aqueous medium at high concentration. Based on the literature data (5,15-21) of binding aroma compounds with different polysaccharides, it is safe to assume that maltodextrins form an inclusion complex with HxAc. As this takes place, some modification of the maltodextrin conformation, (for example, some kind of maltodextrin structurization, through helix formation), occurs on binding with HxAc. According to (77) maltooligosacharides have a ribbonfree



265 like structure: the edges of the ribbon are occupied by polar hydroxyl groups and the flat surfaces are composed of nonpolar patches of the sugar ring faces, so that these patches could form an interior hydrophobic cavity under helix formation. Besides, according to (18) the complexes formed with dextrins should have a helical conformation between 6 and 7 D-glucose monomers per helical turn. Obviously, this process causes the cooperative mechanism of HxAc binding with maltodextrins, whereby H x A c binding with maltodextrins is facilitated with increasing H x A c molecules added to them. In line with this assumption, maltodextrin with the lowest dextrose equivalent (SA-2) exhibits the greater binding capacity regarding HxAc. This could be attributable to the larger degree of molecular polymerization, and thus to the greater readiness of the molecule to undergo structurization, that is helix formation. On the other hand, the cooperative mechanism of HxAc binding with maltodextrins, probably, may be also initiated by an increase in hydrophobicity of the maltodextrin molecules due to an addition of lipophilic HxAc molecules to them.

Effect of Maltodextrin on Binding of HxAc with Legumin in Simple Equimass Mixed Solutions: Maltodextrin + Legumin + Water As a first approximation, let us assume that the binding capacity of maltodextrins does not change in simple mixed solutions with legumin. Bearing this in mind, to consider the effect of the maltodextrins on the legumin binding capacity, we have subtracted the number of moles of HxAc bound by maltodextrin, in accordance with the corresponding binding isotherm, from the total number of moles of H x A c bound to the mixture of legumin with maltodextrin. It is interesting to note that in so doing we have found a totally different binding behavior of legumin when it is in the mixtures with maltodextrins, depending on the D E of these latter. Binding of HxAc with Legumin in the Simple Mixture with Maltodextrin SA-2 Figure 2a compares the binding isotherms of HxAc bound both to legumin alone and legumin in the simple equimass mixture with maltodextrin SA-2. These data suggest a considerable increase in the apparent binding extent of HxAc with legumin under the effect of maltodextrin SA-2. Thus, in the case of the simple mixtures of legumin with maltodextrin SA-2 a positive deviation from the binding behavior of legumin alone was found. The Scatchard plot (Figure 2b), points out the apparent change in the mechanism of HxAc binding with the protein under the effect of maltodextrin SA-2. That is, the binding mechanism varies from HxAc binding on the identical and independent binding sites in the protein globule to the cooperative binding on the interacting binding sites in the presence of maltodextrin SA-2. As a first approximation, the observed effect of maltodextrin on the protein binding capacity relative to HxAc may be attributable to the weak complex formation between protein and polysaccharide in an aqueous medium (9). To gain greater insight into the validity of this assumption, we have attempted to elucidate the nature of interaction between legumin and maltodextrin in an aqueous medium by measurement both of the values of cross second virial coefficient A . i (that characterizes the nature and the intensity of pair biopolymer interactions in the bulk p r


p o

266 a)

b)

( /[L

20-r

v

30


V

] ) x l O - (mol ) 1

f r e e

T

104-

Figure 2. Binding of hexyl acetate with legumin alone and in the presence of maltodextrin SA-2 in an aqueous medium (pH 7.2, ionic strength 0.05 mol/dm ): · , legumin alone; Π, legumin in the mixture with maltodextrin SA-2 a) Binding isotherms: binding extent ν is plotted as a function of the logarithm of free ligand concentration in the system, log [Lfree]- b) Scatchard plot : the ratio of binding extent to free ligand concentration, v/[Lf ], is plotted against binding extent v. 3

ree

aqueous medium (22)) by laser light scattering and the enthalpy of leguminmaltodextrin interactions by mixing calorimetry. Table I shows the values of thermodynamic parameters found. The negative values of the cross second virial coefficient were found for both legumin-maltodextrin pairs studied. Thus the net attractive (thermodynamically favorable) interactions between legumin and maltodextrins were detected in the bulk aqueous medium under the experimental conditions. What is more the values of enthalpy of interaction between legumin and maltodextrins show an exothermic effect. This experimental fact could be attributed to the formation of new bonds between protein and polysaccharide in an aqueous medium. Probably, the exothermic effect found suggests the formation of multiple hydrogen bonds between hydroxyl groups of maltodextrins and the functional groups of protein molecules in an aqueous medium (23). The lack of dextrose equivalent dependence of the thermodynamic parameters of interaction was revealed. Hence, the combined data of light scattering and mixing calorimetry suggest net attractive interactions that are most likely due to the formation of multiple hydrogen bonds between legumin and maltodextrins in the bulk aqueous medium. This weak complex formation is liable to shift the fine hydrophilic-lipophilic balance in the protein


267

Table I. Thermodynamic Parameters of the Interaction between Legumin and Maltodextrins in an Aqueous Medium*


Biopolymer Pair

A pr-pol X 3

10 2

(m mol kg' )

a

ΔH (JgY at Cjf= 5 wt% in the equimass mixture

Legumin Maltodextrin SA-2

-0.29

- 0.46

Legumin Maltodextrin SA-6

-0.24

- 0.23

a

Estimated experimental error ± 10 % . * (pH 7.2, ionic strength 0.05 mol/dm , 20 °C) 3

globule in such a way that some modification of the structure of binding sites in the interior of the protein globule occurs. In response to this modification, the change of the binding mechanism occurs in the presence of maltodextrin SA-2. On the other hand, from the binding data it is also apparent that if, in turn, we subtract the number of moles of HxAc bound by the protein, in accordance with the corresponding binding isotherm, from the total number of moles of H x A c bound to the equimass simple mixture of legumin with maltodextrin SA-2, then we can also find a positive deviation from the binding of HxAc with maltodextrin alone. This experimental fact, not to mention the probable complex formation between macromolecules, can result from an intensification of maltodextrin structurization/helix formation in the presence of legumin, but it is highly improbable. It is very difficult to imagine the mechanism by which legumin could intensify the structurization/helix formation of maltodextrin in an aqueous medium. In summary it may be said that, from the observed data, further structural and thermodynamic elucidation at the molecular level is required to understand the binding behavior of an aroma compound with a simple mixture of legumin with maltodextrin. Binding of HxAc with Legumin in the Simple Mixture with Maltodextrin SA-6 The binding behavior of HxAc to the simple equimass mixture of legumin and maltodextrin SA-6 was also studied. Simple subtraction of the number of moles of H x A c bound by maltodextrin SA-6, in accordance with the corresponding binding isotherm, from the total number of moles of HxAc bound to the equimass mixture showed minor or negative binding of HxAc with legumin. As this takes place, it is very difficult to imagine, on experimental grounds (see Figure la), that maltodextrin SA-6 could be a competitor with legumin for binding of HxAc. B y contrast, legumin can be a real competitor with maltodextrin SA-6 in their equimass mixture regarding the binding of HxAc. In such a manner, legumin could, probably, exclude binding of H x A c with maltodextrin SA-6. We have tested this hypothesis using an approximation that only legumin binds HxAc in the simple equimass mixtures with


268


maltodextrin SA-6. Figures 3a and 3b show a comparison of the binding behavior of legumin alone and in the simple mixture with maltodextrin SA-6. Both the binding isotherms (Figure 3a) and Scatchard plots (Figure 3b) make it clear that the binding behavior of legumin is identical with both samples. (See more details on H x A c binding with the protein in the appropriate chapter of this book). Consequently, legumin is actually a real competitor for maltodextrin SA-6, that is a maltodextrin with higher dextrose equivalent, or with lower molecular weight, relative to binding of H x A c .

Figure 3. Binding of hexyl acetate with legumin alone and with a simple mixture of legumin and maltodextrin SA-6 in an aqueous medium (pH 7.2, ionic strength 0.05 mol/dm ): Φ, legumin alone; V, the mixture of legumin with maltodextrin SA-6. a) Binding isotherms: binding extent ν is plotted as a function of the logarithm of free ligand concentration in the system, log [Lf ]. b) Scatchard plot: the ratio of binding extent to free ligand concentration, v/ [Lj ], is plotted against binding extent v. 1

ree

ree

Effect of the Conjugate Formation between Legumin and Maltodextrin on the Binding Capacity of Legumin in Reference to HxAc A consideration of the binding behavior of the conjugates of legumin with different maltodextrins points to the very different binding behavior of conjugates of legumin with maltodextrin SA-2 and legumin with maltodextrin SA-6.



269 Binding of HxAc with the Conjugate of Legumin with Maltodextrin SA-6 Just before giving an estimation of the data found we should call attention to the exact weight ratio of the protein to maltodextrin in the conjugate. By this means, in the case of the conjugate of legumin with maltodextrin SA-6 this weight ratio (protein : polysaccharide) was 1 : 2. As a first approximation, we assumed that the binding capacity of maltodextrin SA-6 did not change in the conjugate with legumin. In so doing, in studies of the effect of maltodextrin on the binding capacity of legumin in the conjugate, we have subtracted the number of moles of HxAc bound by the maltodextrin, in accordance with the corresponding binding isotherm, from the total number of moles of HxAc bound to the conjugate. Figure 4a shows the binding isotherm of HxAc with legumin alone and then to the conjugate with maltodextrin SA-6. The binding isotherms show a drastic increase in the protein binding capacity when conjugated with maltodextrin SA-6. a)

b)

Figure 4. Binding of hexyl acetate with legumin alone and in the conjugate with maltodextrin SA-6 in an aqueous medium (pH 7.2, ionic strength 0.05 moll dm ): · , legumin alone; V, legumin in the conjugate with maltodextrin SA-6. a)Binding isotherms: binding extent ν is plotted as a function of the logarithm of free ligand concentration in the system, log [Lj ]. b) Scatchard plot: the ratio of binding extent to free ligand concentration, v/ [Lj ], is plotted against binding extent v. 1

rec

rec

As this takes place, the mechanism of binding of HxAc with the protein does not change qualitatively in the conjugate as suggested by Scatchard plots (Figure 4b), namely, the binding of HxAc with legumin in the conjugate also occurs on



270 independent and identical binding sites. Moreover, the Scatchard plots are evidence for the existence of the same total number of binding sites both in the interior of legumin molecules alone and in the conjugate. However, the slopes of the Scatchard plots, differ markedly, pointing to a sharp distinction between the intrinsic binding constants for legumin in the conjugate and alone. Table II gives the values of the characteristic parameters of binding of HxAc both with legumin in the conjugate and alone. The total number of binding sites in the interior of protein molecule is unchanged on the conjugate formation and remains equal to 13. However, the great increase in the intrinsic binding constant suggests a wide variation of structure of binding sites and hence of the protein affinity for HxAc with the formation of conjugate. Indeed, the differential scanning calorimetry data indicate a decrease in the enthalpy of legumin denaturation in the conjugates with maltodextrins, thereby suggesting partial unfolding of the legumin globule in the conjugates (P). If we subtract the number of moles of HxAc bound by legumin (in accordance with the corresponding binding isotherm) from the total number of moles of H x A c bound to the conjugate a positive deviation from the binding of HxAc with maltodextrin SA-6 alone is found, as i f an increase in the binding capacity of maltodextrin SA-6 appears in the conjugate with legumin. The binding behavior of the maltodextrin in the conjugate of this type may be attributable to either our mistaken assumptions that the binding capacity of legumin does not change under the conjugate formation or due to an intensification of the maltodextrin structurization as a result of the covalent binding with legumin. However, it is evident that an answer to this question requires further structural and thermodynamic investigations.

Table II. The Characteristic Parameters of Binding of HxAc with Legumin in the Conjugates with Maltodextrins and Alone (pH 7.2, ionic strength 0.05 mol/dm , 20 °C) 3

Biopolymer

Legumin Conjugate of legumin with maltodextrin SA-6 Conjugate of legumin with maltodextrin SA-2

Total number of binding sites, η

Intrinsic binding Gibbs free energy of binding, constant, AG (kcal/mol) KxIO' (mo ) 3

1

13

1.4

-4.2

13

6.4

-5.1

5

43.4

-6.2

Binding of HxAc with the Conjugate of Legumin with Maltodextrin SA-2 Concerning the binding behavior of the conjugate of legumin with maltodextrin SA-2, it should be noted that the weight ratio of the protein to the maltodextrin in the conjugate solutions, was 1 : 6 . The simple subtraction of the number of moles of H x A c bound by maltodextrin SA-2 (in accordance with the corresponding binding



271

isotherm) from the total number of moles of HxAc bound to the conjugate shows minor or negative binding of HxAc with legumin. This result may be attributed either to the rather high binding capacity of maltodextrin SA-2 in the conjugate or the existence of some structural restrictions on binding of HxAc with the protein coupled with an excess molecules of rather high-molecular weight maltodextrin SA-2 in the conjugate, as i f maltodextrin SA-2 can prevent completely binding of H x A c with legumin in their conjugate. We have tested this supposition using an approximation that only maltodextrin SA-2 binds HxAc in the conjugate. As this takes place, we have compared the binding of HxAc with the conjugate and maltodextrin SA-2 alone. Figure 5 shows the binding data calculated. A comparison of the binding isotherms of the conjugate and maltodextrin SA-2 suggests a pronounced decrease in H x A c binding with the conjugate (Figure 5a). It follows from these results that a marked modification of the maltodextrin molecular structure occurs on the conjugate formation in this case. In line with this assumption, the a)

b)

( /[L

20-r

v

24

V

f r e e

])xlO"

1

(mol")

x

104,

Figure 5. Data on binding of hexyl acetate with maltodextrin SA-2 alone and with the conjugate of maltodextrin SA-2 with legumin in an aqueous medium (pH 7.2, ionic strength 0.05 mol/dm ): •, maltodextrin SA-2 alone; Ώ, the conjugate of maltodextrin SA-2 with legumin. a) Binding isotherms: binding extent ν is plotted as a function of the logarithm offree ligand concentration in the system, log [Lf J. b) Scatchard plot: the ratio of binding extent to free ligand concentration, v/[Lf ], is plotted against binding extent v. 3

K

Ke



272 mechanisms of binding of HxAc with maltodextrin SA-2 alone differs radically from that for the conjugate as shown by Scatchard plots (Figure 5b). In the case of the conjugate, binding of HxAc occurs on the independent and identical binding sites, that is similar to the case of the binding with legumin alone, but that is different from the binding behavior of maltodextrin SA-2 alone, when the cooperative binding mechanism on the interacting binding sites was found (Figure lb). The observed data suggest, that maltodextrin SA-2 coupled covalently with legumin loses its ability to structurization under binding of HxAc. In turn, legumin also changes dramatically its binding capacity in the conjugate as a result of the covalent binding with an excess molecules of high molecular weight maltodextrin. Table II shows a twofold decrease in the number of binding sites in the conjugate and a considerable increase in their affinity for HxAc as compared with the native legumin. This suggests the occurrence of the binding sites with the unique structure under the covalent coupling of maltodextrin SA-2 with legumin. The revealed differences between the effects of maltodextrins with different dextrose equivalent on the protein binding capacity in the conjugate are evidently determined by the molar ratio of maltodextrin to the protein in the conjugates formed. A lower weight ratio of maltodextrin to protein and consequently the lower number of the maltodextrin molecules coupled with legumin, in the case of the conjugate of legumin with maltodextrin SA-6, entails the more fine effect of the maltodextrin on the protein binding capacity for HxAc.

Conclusions 1.

2.

3. 4.

5.

In the simple equimass mixtures of legumin with maltodextrin SA-2 the observed binding data indicate a pronounced increase in the apparent binding extent of H x A c with legumin which is attended with an apparent dramatic change in the binding mechanism of HxAc with the protein In the simple equimass mixtures of legumin with maltodextrin SA-6 the protein can prevent completely the HxAc binding with maltodextrin SA-6, so that, legumin is indeed a real competitor with maltodextrin SA-6 for HxAc. The positive deviation from the binding of HxAc with both maltodextrin SA-6 and legumin was found for their covalent conjugates. The dramatic decrease in the binding of HxAc with the conjugate of legumin with maltodextrin SA-2 was found as compared to the binding behaviors both of legumin and the maltodextrin. A l l experimental findings suggest a profound effect of maltodextrins on the binding capacity of protein, which, evidently, requires further structural and thermodynamic systematic investigations.

Acknowledgement The authors are most grateful to Nestle Research Centre (Switzerland) for the financial support of this research.


273

References


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