Effect of temperature on in vitro ochratoxin A biosorption onto yeast cell wall derivatives

Process Biochemistry 40 (2005) 3008–3016 www.elsevier.com/locate/procbio

Effect of temperature on in vitro ochratoxin A biosorption onto yeast cell wall derivatives Diana Ringot a,*, Benoit Lerzy a, Jean Paul Bonhoure a,b, Eric Auclair c, Eric Oriol d, Yvan Larondelle e a

Institut Supe´rieur d’Agriculture de Beauvais, Rue Pierre Waguet, BP 30313, Beauvais, Cedex 60026, France b Centre de Valorisation des Glucides, 33 Avenue Paul Claudel, 80480 Dury, France c Lesaffre Feed Additives, 1 Rue du Haut Touquet, 59520 Marquette-Lez-Lille, France d Biospringer, 103 rue Jean Jaure`s, 94700 Maison Alfort, France e Universite´ catholique de Louvain, Unite´ de biochimie de la nutrition, Croix du Sud 2/8, 1348 Louvain-la-Neuve, Belgium Received 13 October 2004; received in revised form 10 December 2004; accepted 8 February 2005

Abstract In vitro biosorption of ochratoxin A (OA) onto three yeast industry products: a vinasse containing yeast cell walls (EX16), a purified yeast beta glucan (BETA) and a yeast cell wall fraction (LEC), has been studied as a function of the temperature. Equilibrium binding assays were performed from 4 to 37 8C. The best models for OA biosorption onto EX16, BETA and LEC were identified as respectively Hill’s, Freundlich and Brunauer-Emmett-Teller models. LEC was the most effective adsorbent for OA removal in aqueous solutions. All biosorption processes were spontaneous (negative DG0) and exothermic (negative DH0). For EX16 and BETA, the favourable enthalpic contributions (negative DH0 values) to OA binding, were associated to unfavourable entropic contributions (negative DS0 values). By contrast, the entropic contribution to OA binding was favourable (positive DS0 values) in the case of LEC. The calculated values of the heat capacity (DCp) were close to zero, indicating the absence of a temperature dependency for DH and DS. The results suggest that the remarkable OA biosorption onto LEC involves both polar and non-polar non-covalent interactions and the concomitant reorganization of the water molecules of the solvent. # 2005 Elsevier Ltd. All rights reserved. Keywords: Ochratoxin A (OA); Biosorption; Yeast cell wall derivatives; Isotherms; Thermodynamic parameters

1. Introduction Ochratoxins are a group of secondary metabolites that are produced by some toxic fungi, such as Penicillium verrucosum and Aspergillus ochraceus. The latter fungal species is also referred to as A. alutaceus Berkely et Curtis [1]. Ochratoxin A (OA) is the main mycotoxin in the group of Ochratoxins and it appears to be the only one of toxicological significance. OA has been shown to be a potent nephrotoxic, hepatotoxic, teratogenic, immunotoxic and carcinogenic compound in several animal species [2]. The genotoxic status of OA is still controversial because contradictory results were obtained in various microbial and mammalian * Corresponding author. Tel.: +33 344 062517; fax: +33 344 062526. E-mail address: [email protected] (D. Ringot). 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.02.006

tests [3], notably regarding the formation of DNA adducts [4,5]. OA is considered to be involved in Balkan Endemic Nephropathy (BEN), a severe kidney pathology, occurring in some areas of South-Eastern Europe [6,7]. A complete review on the OA impact on human health has been recently published by Benford et al. [8]. OA contains an isocoumarin moiety linked by a peptide bond to phenylalanine (Fig. 1). Physical data have been published by Pohland et al. [9]. For the toxicokinetics, the weak acidic character of OA is important. The pKa values are in the range 4.2–4.4 and 7.0–7.3, respectively for the carboxyl group of the phenylalanine moiety and the phenolic hydroxyl group of the isocoumarin part [10,11]. This indicates that, in aqueous solutions near the physiological pH, both the monoanion (OA
) and the dianion (OA2
) are present, whereas the fully protonated toxin (OA0) is mainly present in acidic solutions, such as in the upper part of the

D. Ringot et al. / Process Biochemistry 40 (2005) 3008–3016

Fig. 1. Chemical structure of OA.

gastrointestinal tract. OA is rapidly absorbed from the gastrointestinal tract. It is passively absorbed and can undergo secretion and reabsorption through enterohepatic circulation [12]. In addition, reabsorption of OA occurs in the kidney proximal and distal tubules [13]. In blood, OA is bound at more than 99% to serum proteins (mainly albumin), which partly explains its long half-life in the body [14,15]. Renal elimination of the toxin contributes to at least 50% of its total clearance [16]. In mammalian species, OA is hydrolysed in ochratoxin a and phenylalanine by carboxypeptidase A and by some micro-organisms of the gastrointestinal tract [17]. In hepatic cells, it is hydroxylated primarily to 4-R-hydroxyochratoxin A (4-R-OH OA), and to a lesser extent to 4-S-hydroxyochratoxin A (4-S-OH OA) by the mixed function oxydases [18,19]. Three major mechanisms are involved in the toxic effects of OA: inhibition of ATP production, inhibition of protein synthesis and promotion of membrane peroxydation [20]. The occurrence of OA in food and feed has been reported worldwide [21]. It is generally found in cereals, oleaginous seeds, cocoa, green coffee, wine and beer. The intake of OA through contaminated feed may lead to the presence of residues in the products of animal origin (pigs, poultry) like kidney, liver and to a lesser extent muscle, adipose tissue and eggs [22]. Thus, vegetable and animal products can both contribute to the OA contamination of humans. OA is only partially degraded during the technological treatments, which implies that the manufactured commodities may still contain an appreciable quantity of OA. Different approaches are developed in order to reduce the adverse effects of mycotoxins. The most applied method to prevent mycotoxicosis in animals consists in the addition of adsorbents to animal feed, in order to bind the mycotoxins in the gastro-intestinal tract. Adsorption onto various types of compounds (hydrated sodium calcium aluminosilicate, HSCAS, kaolin, silica binding agent, bentonite, etc.) has been extensively studied in recent years [23,24]. Silicates are the most widespread commercially available feed additives for mycotoxins binding [25]. Yeasts and yeast cell wall products have also been suggested to reduce aflatoxicosis in poultry [26,27]. Efficient in vitro adsorption of zearalenone on the modified yeast glucan and yeast cell walls are also recently reported [28,29]. Concerning OA adsorption, different binding agents including activated carbons [30], zeolites [31], diatomaceous earth [32], cholestyramine and mixtures of sterilized yeast and fermentation residua of beer production [25] were

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reported to remove OA in vitro. Castellari et al. [33] examined several fining treatments to reduce OA levels of red wine. They found that potassium caseinate and activated carbon were the best agents to remove OA. Yeast biomass may be regarded as a good source of adsorbent material, due to the presence in their cell wall of some specific macromolecules, such as the mannoproteins and beta glucans. OA biosorption on yeast industry-derived products was therefore investigated and from 32 to 98% of adsorption was found depending on the material used and toxin concentration [34]. The current in vitro study presents a thermodynamical approach to characterize the binding of OA onto three yeast products. Isotherm analysis at three temperatures (4, 25 and 37 8C) was used to determine the following parameters: Gibbs free energy (DG0), enthalpy (DH0) end entropy (DS0) of OA adsorption. This allowed the identification of the best equilibrium adsorption model for each adsorbent, to select the best potential adsorbent and to suggest some hypotheses on the mechanism of OA yeast cell wall biosorption.

2. Materials and methods The yeast-derived products tested as biosorbents were obtained from Bio-Springer, Maison Alfort, France. Two of these adsorbents, EX16 (a vinasse containing 16% of liquid yeast cell walls) and LEC (a dry yeast cell wall fraction) are industrial by-products. The third adsorbent, BETA, is a purified beta glucan fraction of yeast cell walls. Ochratoxin A was purchased from Sigma Chemical Company (St. Louis, MO, USA). A primary stock solution (200 mg/L) was prepared in methanol. Mycotoxin test solutions (0.5, 1.0, 2.0, 5.0 and 10.0 mg/L) were prepared by dilution of the methanolic stock solution in deionised distilled water. Samples of 500 mg of adsorbents were placed in screwcap test tubes with 10 mL of the mycotoxin test solutions. Two sets of controls were prepared, one corresponding to 10 mL of mycotoxin test solutions without the addition of adsorbent, and another one consisting of 500 mg adsorbent in 10 mL of deionised distilled water. The controls and test tubes were placed on a horizontal shaker-incubator at 400 rpm. Preliminary investigations showed that equilibrium uptake was attained rapidly with practically no change observed after a period of 30 min. All adsorptions were run for 90 min to ensure complete uptake. Three temperatures were studied: 4.0  1.0, 25.0  1.0 and 37.0  1.0 8C. After the incubation period, solid particles (adsorbent) were separated from the supernatant by centrifugation at 6000  g for 20 min at respectively, 4, 25 and 37 8C in an ALC 4239 R centrifugation system (Fisher Scientific Labosi, Elancourt, France). All experiments were carried out in triplicate.

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D. Ringot et al. / Process Biochemistry 40 (2005) 3008–3016

The supernatant volume was measured, and subsequently mixed with an identical volume of methanol: deionised distilled water mixture (50:50). The mixture was then diluted, filtered and analysed by HPLC. The chromatographic system consisted of a SpectraSYSTEM1 P1500 isocratic pump (Thermo Separation Products, Fremont, CA, USA) equipped with a AS 3000 model injection valve (100 mL) (Thermo Separation Products), a Waters 2475 Multi l-fluorescence detector (Waters, Milford, MA, USA) equipped with a 150 W xenon lamp (lexcitation = 333 nm and lemission = 470 nm) and a PC1000 integration software (Thermo Separation Products). The analytical column was an Alltima reversed-phase column C18 (25 cm  4.6 mm i.d., 5 mm particles), preceded by an Alltima C18 precolumn (7.5 mm  4.6 mm i.d., 5 mm particles) (Alltech, Templemars, France). The columns were left at room temperature. The mobile phase was made of a mixture of HPLC grade acetonitrile:water:acetic acid (45:54:1), filtered through a 0.22 mm filter membrane, degassed and used at a flow-rate of 1.0 mL/min. Retention time of OA was 7.5 min. The quantity of OTA adsorbed was determined by the following equation: Qeq ¼

ðC0
Ceq ÞV ; m

(1)

Qeq, quantity of OA adsorbed per gram of adsorbent (mg/g); C0, initial concentration of OA in solution (mg/L); Ceq, residual toxin concentration at equilibrium (mg/L); V, volume of solution (L); m, mass of adsorbent (g). The highest standard deviations calculated for the OA concentration measurements were 0.368, 0.598 and 0.036 mg/L, respectively for EX16, BETA and LEC experiments.

3. Results and discussion 3.1. Adsorption isotherms The effect of temperature on the adsorption behaviour of OA onto three yeast-derived products was studied in the range 4–37 8C. Equilibrium adsorption data, commonly known as adsorption isotherms, are important in the characterisation of the adsorption phenomenon. Adsorption equilibrium is established when the quantity of the toxin being adsorbed (Qeq) is equal to the quantity being desorbed. Then, the equilibrium concentration in solution (Ceq) remains constant. The plots Qeq = f(Ceq) for all adsorption experiments are presented in Fig. 2. For each of the three adsorbents studied, three temperatures were considered 4, 25 and 37 8C. OA binding onto EX16 reached 34–49% at 4 8C, 32–43% at 25 8C and 22–30% at 37 8C. The binding ability of EX16 thus

Fig. 2. Plot of adsorbed amount of OA (Qeq) onto EX16 (A), BETA (B) and LEC (C) vs. equilibrium solute concentration (Ceq) at various temperatures.

decreases when the temperature increases from 4 to 37 8C, which suggests that the biosorption of OA onto EX16 is an exothermic process. OA removal by beta glucans was 54–59% at 4 8C, 37– 51% at 25 8C and 37–50% at 37 8C. The decrease in sorption ability with the increase in temperature suggests, once again, an exothermic biosorption process. LEC was a very good adsorbent able to bind 96–100%, 95–100% and 94–100% of OA added at respectively 4, 25 and 37 8C. Since OA is almost entirely adsorbed on LEC in the range of toxin concentrations studied and at all tested temperatures, the thermic effect of the biosorption process is difficult to assess in this case. 3.2. Isotherm analysis To characterize the adsorption isotherms, four classical isothermal models was used, namely the Freundlich, Langmuir, Hill and BET models, in their linear forms. 3.2.1. Freundlich empirical model The empirical Freundlich [35] equation based on sorption onto a heterogeneous surface is given below by Eq. (2). The linearized form of Freundlich equation is given in Eq. (3).

D. Ringot et al. / Process Biochemistry 40 (2005) 3008–3016 1=n Qeq ¼ Kf  Ceq

ln Qeq ¼ ln Kf þ

(2) 1 ln Ceq n

(3)

Ceq, residual toxin concentration at equilibrium (mg/L); Kf, Freundlich adsorption constant (mg/g)(mg/L)n; n, Freundlich adsorption constant; Qeq, adsorbed toxin quantity per gram of biomass (mg/g); Kf and 1/n are indicators of adsorption capacity and adsorption intensity, respectively. A plot of ln(Qeq) versus ln(Ceq) should indicate a straight line of slope 1/n and intercept ln Kf. 3.2.2. Langmuir model The Langmuir [36] model is valid for monolayer sorption to a surface with a finite number of identical sites. The Langmuir model is based on four basic assumptions [37]: -

Sorbate is adsorbed at a fixed number of well defined sites, Each site can hold one sorbate molecule, All sites are energetically equivalent, There is no interaction between molecules adsorbed on neighbouring sites.

The well-known expression of the Langmuir model is given by Eq. (4), where Qeq (mg/g) and Ceq (mg/L) are the amount of adsorbent per unity of biomass weight and the unadsorbed toxin concentration in solution at equilibrium, respectively. Qmax (mg/g) is the maximum amount of toxin per unit of biomass weight to form a complete monolayer and KL (L/mg) is a constant related to the affinity of the binding sites. The linearized form of Langmuir equation is presented in Eq. (5). Qeq ¼

Qmax KL Ceq 1 þ KL Ceq

1 1 1 1 ¼  þ Qeq KL Qmax Ceq Qmax

(4)

(5)

A plot of Ceq/Qeq versus Ceq should indicate a straight line of slope 1/Qmax and intercept 1/KLQmax. When the equilibrium concentration, Ceq, is low, 1  KLCeq and Langmuir becomes Henry’s law (Qeq = KHCeq). 3.2.3. Brunauer-Emmett-Teller (BET) model BET isotherm [38] is the theoretical model for multilayer adsorption. It is the most widely applied model in studies of gas–solid equilibrium. This model assumes multilayer adsorption and was developed to describe adsorption phenomena in which successive molecular layers of adsorbate form beyond the completion of a monolayer. The extension of this model to liquid/solid interface is described by Eq. (6), which is linearized in Eq. (7).

Qeq ¼

3011

Q0max CBET Ceq ðCs
Ceq Þ  ½1 þ ðCBET
1ÞCeq =Cs Þ

(6)

Ceq 1 CBET
1 Ceq ¼ 0 þ 0  Qeq ðCs
Ceq Þ Qmax CBET Qmax CBET Cs

(7)

CBET, BET adsorption constant relating to the energy of interaction with the surface (L/mg); Ceq, residual toxin concentration at equilibrium (mg/L); Qeq, adsorbed toxin quantity per gram of biomass (mg/g); Q0max , maximum specific uptake corresponding to monolayer saturation (mg/g); Cs, saturation concentration of the solute corresponding at monolayer saturation (mg/L). The plot of Ceq/[Qeq(Cs
Ceq)] versus Ceq/Cs permits the calculation of CBET and Q0max . 3.2.4. Hill model The Hill equation [39] can be used to describe the binding of different species onto an heterogeneous substrate. This model assumes that the adsorption is a cooperative phenomenon where the ligand binding at one site on a macromolecule influences ligand binding at a different site on the same macromolecule. The Hill equation is given in Eq. (8), which is linearized in Eq. (9). Qeq ¼

nH Qmax Ceq

(8)

nH KD þ Ceq   Qeq ln ¼ nH ln Ceq
ln KD Qmax
Qeq

(9)

Ceq, residual toxin concentration at equilibrium (mg/L); Qeq, adsorbed toxin quantity per gram of biomass (mg/g); KD, Hill constant, KD ¼ KdnH ; Kd, dissociation constant per site (mg/L); Qmax, maximum specific uptake corresponding to sites saturation (mg/g); nH, Hill cooperativity coefficient of the binding interaction (nH > 1, positive cooperativity; nH = 1, no cooperativity; nH < 1, negative cooperativity). Since formerly the magnitude of R2 was confirmed to be a correct indicator for relative quality of fit of the linear isotherms [34], the analysis was restricted to the linear equations in the present paper. The values of linear coefficients are presented in Tables 1–4. Table 1 Freundlich parameters for OA biosorption onto EX16, BETA and LEC at various temperatures Adsorbent

Temperature (8C)

R2

KF (mg/g)

n (mL/g)

EX16

4 25 37

0.954 0.974 0.986

0.014 0.013 0.008

1.01 1.04 1.07

BETA

4 25 37

0.997 0.961 0.991

0.027 0.016 0.015

1.04 1.03 1.00

LEC

4 25 37

0.872 0.896 0.919

0.244 0.202 0.185

2.14 2.05 2.10

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D. Ringot et al. / Process Biochemistry 40 (2005) 3008–3016

Table 2 Langmuir parameters for OA biosorption onto EX16, BETA and LEC at various temperatures Adsorbent

Temperature (8C)

R2

Qmax (mg/g)

KL (L/mg)

EX16

4 25 37

0.979 0.982 0.985

0.136 0.150 0.171

0.155 0.109 0.05

BETA

4 25 37

0.989 0.920 0.954

0.275 0.134 0.117

0.109 0.169 0.176

LEC

4 25 37

0.971 0.981 0.977

0.117 0.109 0.100

17.438 12.938 12.722

Table 3 BET parameters for OA biosorption onto EX16, BETA and LEC at various temperatures Adsorbent

2

Q0max (mg/g)

CBET (L/mg)

0.860 0.867 0.890

0.015 0.015 0.021

56.606 26.036 6.779

4 25 37

0.997 0.915 0.897

0.049 0.019 0.028

4.161 9.912 5.944

4 25 37

0.999 0.997 0.998

0.044 0.045 0.046

23.327 22.750 20.090

Temperature (8C)

R

EX16

4 25 37

BETA

LEC

For BETA, two models (Freundlich and BET) fit very well with the experimental data (R2 = 0.997) at 4 8C. At 25 and 37 8C however, the highest values of R2 were obtained only with the Freundlich model. The higher adsorption capacity, Kf, at 4 8C indicates that the phenomenon is favoured at lower temperature. Since the values of nF in the Freundlich model were very close to 1, the OA biosorption on BETA can be considered a linear solid/liquid partition, which follows Henry’s law. The Langmuir model can be used to predict whether a sorption system is favourable or unfavourable. This is done through a dimensionless constant, KR, which is defined by the following equation [41]. KR ¼

1 1 þ KL C0

(10)

For favourable adsorption, the KR value should fall in the range 0 to 1. The adsorption is considered unfavourable when KR > 1, the isotherm is linear when KR = 1 and the adsorption is irreversible when KR = 0. In this study, the values of KR for OA biosorption onto all yeast by-products are comprised between 0 and 1, which suggests a favourable process for all systems. The KR values have been calculated for EX16, BETA and LEC in the range of 0.387–0.976, 0.378–0.934 and 0.005–0.146, respectively (data not shown). The very low KR values obtained for LEC indicate an almost irreversible adsorption phenomenon. 3.3. Thermodynamic parameters

For EX16, the best coefficients of determination R2 were obtained with the Hill’s model for all temperatures studied. OA adsorption on EX16 can thus be considered as a cooperative phenomenon. The value of the Qmax decreases when the temperature increases because of an increase in desorption [40]. For LEC, the best model was BET, according to the R2 values. OA adsorption onto LEC is thus a multilayer phenomenon. The quantity of OA needed to fill the first monolayer, Q0max , slightly increased (0.044–0.046 mg OA per gram of LEC) with the temperature. Table 4 Hill’s parameters for OA biosorption onto EX16, BETA and LEC at various temperatures Adsorbent

Temperature (8C)

R2

Qmax (mg/g)

nH

EX16

4 25 37

0.984 0.996 0.994

0.093 0.078 0.070

1.510 1.470 1.430

4.011 4.054 5.060

BETA

4 25 37

0.989 0.940 0.987

0.430 0.174 0.164

1.040 1.040 1.180

14.440 8.004 8.935

LEC

4 25 37

0.944 0.964 0.981

0.888 1.892 2.950

0.768 0.762 0.710

1.640 4.854 9.927

KL (L/mg)

In adsorption studies, both energy and entropy factors must be considered in order to determine which processes will occur spontaneously. The Gibbs free energy change, DG0 (kJ/mol), is the fundamental criterion of spontaneity and is given by the Gibbs–Helmholtz equation: DG0 ¼
RT ln K0 ;

(11)

where R is the universal gas constant and T is the absolute temperature (K). The equilibrium constant K0 for the adsorption reaction is defined in Eq. (12). K0 ¼

Qeq Ce

(12)

Qeq is the molar OA concentration in adsorbed phase (mol/ kg), Ce is the residual toxin concentration at equilibrium (mol/L), values of K0 are obtained by plotting ln(Qeq/Ce) versus Ce and extrapolating Ce to zero [42,43]. The DG0 values calculated from our experimental data are presented in Table 5. The negative values of DG0 for all adsorption experiments indicate the spontaneous nature of the sorption. For EX16 and BETA, the
DG0 values decrease as temperature increases, which means that an increase in temperature leads to an increase in desorption from the binder surface. By contrast, the value of
DG0

D. Ringot et al. / Process Biochemistry 40 (2005) 3008–3016

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Table 5 Thermodynamic parameters for OA biosorption onto yeast by-products Adsorbent

Temperature (K)

ln K0

DG0 (kJ/mol)

DH0 (kJ/mol)

DS0 (J/mol)

EX16

277 298 310

2.97 2.62 2.11


6.84
6.48
5.45


17.77


39.07

277 298 310

3.31 2.96 2.75


7.62
7.32
7.07


11.95


15.64

277 298 310

6.86 6.69 6.57


15.79
16.57
16.94


6.07

35.13

BETA

LEC

increases with the rise in temperature for LEC, indicating that the sorption process is favoured at higher temperature. In the van’t Hoff equation (Eq. (13)), the constant of equilibrium K0 is expressed in terms of enthalpy (DH0, kJ/ mol) and entropy (DS0, kJ/mol K) changes: ln K0 ¼


DH 0 DS0 þ RT R

(13)

where T is the absolute temperature (K). The van’t Hoff equation considers that enthalpy and entropy changes are independent from temperature. This assumption must however be verified, which can be done using the integrated form of the Gibbs–Helmholtz equation (Eq. (14)), DGT ¼ DH 0
TDS0    T þ DCp T
Tref T
T ln ; Tref T

(14)

where DGT is the free energy at each temperature (kJ/mol), T is the absolute temperature (K), Tref T is the reference temperature chosen arbitrarily, DCp is the heat capacity (kJ/mol K), and DH0 (kJ/mol) and DS0 (kJ/mol K) are the fitted van’t Hoff enthalpy and entropy values. Van’t Hoff assumption is valid only if the DCp values are close to zero. The values of DH0 and DS0 were determined from the slope and the intercept of the plots of ln K0 versus 1/T shown in Fig. 3. The values obtained are presented in Table 5. Based on these values, the DCp values were calculated using Eq. (14). The values obtained are close to zero (
0.073,
0.009 and
0.007 kJ/mol K, respectively for EX16, BETA and LEC), which validates Van’t Hoff’s assumption. The low values of DCp are generally associated with the nonspecific ligand–protein interactions [44]. In the present case, this suggests that only minor conformational changes are needed for toxin adsorption. As presented in Table 5, all DH0 values for OA biosorption onto the EX16, BETA and LEC are negative, confirming the exothermic nature of the phenomenon. The magnitude of the DH0 value gives an indication on the type of adsorption, which can be either physical or chemical. In the first case, the energy requirement is small (<40 kJ/mol),

Fig. 3. Van’t Hoff plot for OA biosorption onto EX16 (A), BETA (B), LEC (C).

allowing the equilibrium to be attained rapidly and the process to be easily reversible [45]. By contrast, the chemical adsorption involves higher enthalpy changes (>40 kJ/mol). In this study, the values of enthalpy were less than 20 kJ, indicating a physical adsorption phenomenon. The negative DS0 for OA biosorption onto EX16 and BETA may be understood in terms of restriction of the movement of the molecules on the surface (two dimensions), as compared to the bulk solution (three dimensions) [46]. The binding of OA on EX16 and BETA is thus only enthalpically driven. Generally, this situation corresponds to polar non-covalent interactions (benefit in enthalpy associated to a cost in entropy), such as electrostatic interactions or hydrogen bonds [47]. OA being mainly in its monoanion (OA
) form at the pH value of the systems employed (close to 5.3), it may easily be involved in polar non-covalent interactions. In the case of LEC, the entropy change DS0 is positive and reflects an increase in the randomness at solid/liquid

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D. Ringot et al. / Process Biochemistry 40 (2005) 3008–3016

interface [48,49,50]. One of the possible explanations for the entropic positive change in the case of OA biosorption onto LEC is the contribution of hydrophobic interactions between the partners. Interestingly, LEC is largely made of mannoproteins of the yeast cell wall in contrast to EX16 and BETA, which are almost protein free [51]. The mannoproteins may contain hydrophobic domains enabling hydrophobic interactions to occur with OA, especially at the pH of the LEC adsorption system (pH 3), where OA is mainly in its non-ionized form. This hypothesis is supported by the microcalorimetry measurements of Doherty et al. [52], which suggested that the binding of a protein to an hydrophobic surface are largely entropically driven. The positive entropy change may also be caused by the decrease in the number of water molecules surrounding the partners upon hydrophobic interaction [53,54], thus increasing the disorder in the system. Generally, hydrophobic interactions are associated to a cost in enthalpy. The reverse is however observed during OA adsorption onto LEC, suggesting that this binding also involves polar non-covalent interactions. Similar observations were reported by Lin et al. [55], who found an enthalpy of adsorption ranging from 0 to
10 kJ/mol in the case of protein binding on the phenyl-derived surfaces, whereas the entropy of binding ranging from about 30 to 80 J/mol K. The values of enthalpy and entropy of OA adsorption on LEC in the present work are comparable with the data of Lin et al. [55], suggesting a possible role of the phenylalanine (Phe) moiety of OA in the phenomenon. The role of the Phe moiety in OA binding on the selective polymers for solidphase extraction has been previously reported by Jodlbauer et al. [56] who found that binding was impaired by the replacement of Phe by structurally related compounds. These authors suggest that the aromatic domain of Phe contributes to the interaction, both through the stabilisation of intermolecular complexes made via electrostatic p–p interactions (face-to-face stacked geometry) and hydrophobic associations (offset stacked geometry). The existence of a multilayer adsorption in the case of OA adsorption onto LEC suggests a predominant role of face-to-face and/or offset stacked geometries. Moreover, the aromatic ring of Phe may also act as acceptor in hydrogen bonds with donor groups, like the amide group (–CONH–) of proteins, in an edge-face stacked geometry [57]. The isocoumarin moiety of OA may also participate in the interaction with LEC. Early studies published by Chu [14,15] already underlined the importance of the isocoumarin part of OA in its binding to bovine serum albumin. Two types of interactions can occur: hydrophobic forces and ionic forces involving the phenolate ion of the toxin. In our study, the phenolic group is however in its protonated form and its participation should thus be limited to hydrogen bonds. Thus the OA binding onto LEC involves both non-polar and polar interactions. The non-polar interactions involve the aromatic rings of OA and hydrophobic amino acids of LEC. The polar interactions can be explained in different

complementary ways: (i) electrostatic ionic interactions involving the carboxylic group of OA and basic amino acids of LEC proteins, (ii) electrostatic p–p interactions involving the aromatic rings of OA and aromatic amino acids of LEC, (iii) hydrogen bonds involving the OA phenol and amide group as donor and acceptor groups of LEC, (iv) hydrogen bonds involving OA aromatic rings as acceptors interacting with donor groups of LEC. The relative importance of each of these interactions in the OA-LEC complexes still remains an open question. Additional research on influence of the amount of the adsorbents and pH conditions on OA adsorption onto yeast cell products will further improve an understanding of this biosorption phenomenon.

4. Conclusions This research was intended to increase an understanding of the mechanisms of ochratoxin A binding onto yeast cell products, on the basis of calculated thermodynamic parameters. The results of the study have led to the following conclusions: (i) the best models for OA adsorption onto EX16, BETA and LEC were, respectively identified as Hill, Freundlich and BET models, (ii) among the three adsorbents studied, the dried yeast cell wall (LEC) is the most effective for OA removal from aqueous solutions, (iii) the negative values of DG0 for all adsorption experiments indicate the spontaneous nature of the sorption, (iv) the negative values of DH0 for OA biosorption onto the EX16, BETA and LEC indicate an exothermic phenomenon and, in addition, their low absolute values designated a physical process, (v) the negative values of DS0 for OA biosorption onto EX16 and BETA indicate a randomness decrease at solid/solution interface, whereas for the toxin biosorption on LEC, the positive value of DS0 suggests hydrophobic interactions and an increase in the degree of freedom of the water molecules in the bulk solution, (vi) the values of DCp near zero indicate that DH and DS are almost independent from the temperature.

Acknowledgement The authors thank Dr. Abalo Chango from ISAB for his scientific interest in this study.

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