Study of interactions between anionic ...

Colloids and Surfaces B: Biointerfaces 167 (2018) 516–523

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Study of interactions between anionic exopolysaccharides produced by newly isolated probiotic bacteria and sodium caseinate Yousra Abid a , Ichrak Joulak a , Chedia Ben Amara b , Angela Casillo c , Hamadi Attia a , Adem Gharsallaoui b , Samia Azabou a,∗ a

Université de Sfax, ENIS, Laboratoire Analyse, Valorisation et Sécurité des Aliments, Sfax, 3038, Tunisia Univ Lyon, Université Claude Bernard Lyon 1, ISARA Lyon, Laboratoire BioDyMIA (Bioingénierie et Dynamique Microbienne aux Interfaces Alimentaires), Equipe Mixte d’Accueil n◦ 3733, IUT Lyon 1, technopole Alimentec, rue Henri de Boissieu, 01000, Bourg en Bresse, France c Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte S.Angelo, via Cintia 4, I-80126, Napoli, Italy b

a r t i c l e

i n f o

Article history: Received 3 January 2018 Received in revised form 7 April 2018 Accepted 24 April 2018 Available online 26 April 2018 Keywords: Probiotic bacteria Exopolysaccharides Sodium caseinate Complex coacervation

a b s t r a c t The present study aims to evaluate the interactions between four exopolysaccharides (EPS) produced by probiotic bacteria and sodium caseinate (Cas) in order to simulate their behavior in dairy products. Complexation between the produced EPS samples and Cas was investigated as a function of polysaccharide to protein ratio. The highest turbidity and average size of complexes were formed at an EPS/Cas ratio of 3 (corresponding to 1 g/L of EPS and 0.33 g/L of Cas) as a result of the combination of individual complexes to form aggregates. Zeta potential measurements and Cas surface hydrophobicity results suggested that complex formation occurred essentially through electrostatic attractions with a possible contribution of hydrophobic interaction for EPS-GM which was produced by Bacillus tequilensis-GM. Afterwards, the effect of pH on the complexation between biopolymers was studied when EPS and Cas concentrations were maintained constant at 1 and 0.33 g/L, respectively. pH was adjusted to 3.0 and 3.5, respectively. Results showed that the highest amount and sizes of EPS/Cas complexes were formed at pH 3.5 and that EPS-GM enabled to obtain the biggest and highest amount of aggregates. Therefore, the obtained results support the fact that the simultaneous presence of EPS and Cas in dairy products results in complexes formation via electrostatic interactions depending on EPS/Cas ratio and pH of the medium. © 2018 Elsevier B.V. All rights reserved.

1. Introduction Polysaccharides and proteins are natural polymers that are often simultaneously present in several food colloids formulations. The texture and the stability of these food systems are generally controlled by the polysaccharide/protein interactions through their thickening and surface properties [1]. It has been reported that the functional properties of each biopolymer alone are generally improved by the interaction with the other biopolymer [2], which is of great interest in new food formulation development. In fact, the formed complexes can be used as meat analogues [3], fat substitutes [4], thickening agents and emulsion stabilizers [5], for food ingredient encapsulation and stabilization of food products [6], or for the development of composite edible films [7]. Protein/polysaccharide complex formation mostly occurs between oppositely charged macromolecules through electrostatic

∗ Corresponding author. E-mail address: [email protected] (S. Azabou). https://doi.org/10.1016/j.colsurfb.2018.04.046 0927-7765/© 2018 Elsevier B.V. All rights reserved.

attractions. Below their pHi , proteins possess positive charges and may interact with anionic polysaccharides having phosphate, carboxylic, or sulfate groups. It has been shown that the formation of these complexes depends on several physicochemical factors such as pH, ionic strength, biopolymer type, protein/polysaccharide ratio and thermal history [8]. By mixing aqueous solutions of polysaccharides and proteins, one may observe either one of the following possibilities: thermodynamic incompatibility, co-solubility or complex coacervation. The resulted complexes can be soluble or contribute to aggregative phase separation [9]. Apart from electrostatic interactions, complex formation can likewise occur through van der Waals forces or hydrophobic interactions [10]. Several works have already studied interactions between different polysaccharides and sodium caseinate (Cas) [11–14]. Cas is an amphiphilic protein that is broadly used to improve texture, shelf life and nutritive value of emulsions and foams [15]. A group of polysaccharides with a particular interest in dairy applications are microbial exopolysaccharides (EPS) acting as potential natural stabilizers and thickeners. These EPS can be produced in situ during fermentation of milk by lactic acid bacteria (LAB) and/or produced

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by probiotic bacteria which were introduced in dairy products [16]. Indeed, various probiotic preparations are currently commercially available, including strains of Bifidobacterium, Lactobacillus and some Bacillus sp. [17,18]. During the fermentation, the amount of EPS produced by these bacteria will gradually increase, the interactions between the biopolymers will continuously change as the concentration of EPS increases, and both attractive and repulsive interactions may occur [19]. Several works were made to study the presence of associative interactions between EPS and milk proteins [20–24]. In the context of the above discussion, we aimed to study the interaction between four different EPS produced by recently isolated probiotic bacteria from spontaneously fermented foods and beverages with sodium caseinate in order to predict their behavior as functional ingredients that may improve texture, mouthfeel, structure and shelf life of the final dairy product. 2. Materials and methods 2.1. EPS production Four probiotic strains identified as Leuconostoc citreum-BMS, Leuconostoc mesenteroides-TMS, Pediococcus pentosaceus-DPS and Bacillus tequilensis-GM, were recently isolated from Tunisian spontaneously fermented bovine and turkey meat sausages (BMS and TMS), date palm sap (DPS) and goat milk (GM), respectively [25]. The isolates were maintained on MRS agar plates (Oxoid) or LB agar plates for B. tequilensis-GM strain, kept at 4 ◦ C under anaerobic conditions and stored at −80 ◦ C in MRS broth or LB broth supplemented with glycerol (v/v: 80/20). The EPS were produced and purified as described by Abid et al. [25]. Briefly, a modified ESM (Exopolysaccharides selection medium) used by van den Berg et al. [26] containing 5% skimmed milk, 0.35% w/v yeast extract, 0.35% w/v peptone and 5% w/v sucrose, was sterilized at 120 ◦ C for 20 min and inoculated (1%) with an overnight culture (OD≈1). After 29 h of incubation at 30 ◦ C, cells were removed by centrifugation (9 000 rpm, 20 min, 4 ◦ C). Afterwards, proteins were removed by adding trichloroacetic acid (TCA) solution to the culture supernatant to give a final concentration of 4% (w/v) and then by centrifugation at 10 000 rpm for 30 min at 4 ◦ C. Two volumes of absolute ethanol were added to the obtained supernatant and kept overnight at 4 ◦ C. Crude EPS was harvested by centrifugation (9000 rpm, 20 min, 4 ◦ C), dissolved in ultrapure water and dialyzed for 2 days at 4 ◦ C using a dialysis membrane (Medicell International, Ltd., London, U.K.) having a cut-off of 12–14 kDa. After dialysis, the solution was lyophilized (Telstar Cryodos equipment, Spain). The carbohydrate content was evaluated by the phenol-sulfuric acid method [27], the protein content was determined by Lowry method [28] and the phosphate content was deduced from inorganic phosphate determination on a 5500 inductively coupled plasma (ICP) instrument (Perkin Elmer). 2.2. Determination of monosaccharide composition EPS samples (0.5 mg) were mixed with 1 mL of HCl/CH3 OH (1.25 M) and subjected to methanolysis for 16 h at 80 ◦ C [29]. Fatty acids were extracted twice with hexane, the methanol layer was dried and acetylated with 25 ␮L of acetic anhydride and 25 ␮L of pyridine and then kept at 100 ◦ C for 30 min. After evaporation of solvents, 1 mL of chloroform and 1 mL of MilliQ water were added to the sample and the mixture was shaken. The organic phase was recovered after centrifugation for 3 min at 1 500 rpm and washed two times with MilliQ water. Finally, it was concentrated and injected into the GC–MS. All the sample derivatives were analyzed on an Agilent Technologies gas chromatograph 6850A

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equipped with a mass selective detector 5973N and a Zebron ZB5 capillary column (Phenomenex, 30 m × 0.25 mm i.d., flow rate 1 mL/min, He as carrier gas). Acetylated methyl glycosides were analyzed using the following temperature program: 140 ◦ C for 3 min, 140 ◦ C → 240 ◦ C at 3 ◦ C/min. 2.3. Molecular weight determination The weight–average molecular weight (Mw ) of the EPS was determined by high-performance size exclusion chromatography (HPLC-SEC, Agilent 1200 Series System, Hewlett-Packard, Germany) with refractive index (RI) detection. The column was eluted with 50 mM NH4 HCO3 , at 35 ◦ C and at a flow rate of 0.8 mL/min [30]. The polymer solutions were prepared at 1 mg/mL and 50 ␮L were injected. The calibration curve was obtained by using dextran standards (1, 50, 150, 270, 410, 670, 1500 kDa) obtained from Sigma-Aldrich. 2.4. EPS and sodium caseinate (Cas) solutions preparation Stock solutions of Cas (0.5 g/L) and EPS (3 g/L) were prepared by dissolving powders in imidazole/acetate buffer (5 mM, pH 3.0) and stirred with a magnetic stirrer until a complete dissolution. The pH was adjusted by adding HCl (1 M) or NaOH (1 M) solution. 2.5. EPS/Cas complex formation and characterization 2.5.1. Effect of EPS/Cas ratio on the complexation Complexes of EPS and Cas were prepared by mixing different weight ratios of the biopolymers in imidazole/acetate buffer (5 mM, pH 3.0). EPS concentrations were 0; 0.2; 0.4; 0.6; 0.8 and 1 g/L and Cas concentration was kept constant at 0.33 g/L. The pH of the suspensions was re-adjusted before each test. 2.5.2. Effect of pH on the complexation The effect of pH variation on the complexation of biopolymers prepared in imidazole/acetate buffer (5 mM) was investigated at both pH 3.0 and pH 3.5. EPS/Cas ratio was kept constant at 3. 2.5.3. Turbidity measurement Suspension turbidity was determined as a function of EPS/Cas ratio and as a function of pH, respectively, by measuring the optical density at 600 nm at 25 ◦ C using a UV/Vis spectrophotometer (Jenway 7305, Villepinte, France) against pure Cas solution (without EPS). 2.5.4. Zeta potential measurement The zeta potential (potential ␨) of biopolymer solutions diluted (0.5% (w/w) in imidazole/acetate buffer (5 mM) and adjusted to the suitable pH was determined using a Zetasizer NanoZS90 (Malvern Instruments, Malvern, UK). 2.5.5. Particle size measurement Particle size measurement of EPS/Cas complexes formed at constant ratio and various pH (3.0 and 3.5) was performed using a laser diffraction instrument (Malvern Mastersizer 3000, UK). Before measurements, the complexes were diluted with imidazole/acetate buffer (5 mM) adjusted to the suitable pH in order to avoid multiple scattering effects. The complexes were continuously stirred throughout the measurement to ensure that the samples were homogeneous. The volume particle diameter (D [4,3]) was calculated according to the following equation: D [4, 3] =





Di4 /

Di3

Di : the diameter of class “i” [31].

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Table 1 EPS characterization.

EPS-BMS EPS-TMS EPS-DPS EPS-GM

Monosaccharide composition

Molecular weight (Da)

Sugar content (%)

Phosphate content (%)

Glucose, Mannose, fructose Glucose, mannose, fructose Glucose, Mannose, fructose Fructose, Glucose, Mannose, Galactose, Galactosamine, N-acetyl-glucosamine

1.8 106 1.8 106 1.2 106 1.8–2.5 106

90.4 ± 0.50 89.4 ± 0.42 93.5 ± 0.74 95.6 ± 0.10

0.56 ± 0.03 0.58 ± 0.02 0.32 ± 0.01 1.02 ± 0.04

2.5.6. Measurement of surface hydrophobicity The protein surface hydrophobicity (S0 ) was measured spectrofluorometrically using 1-aniline-8-naphthalene sulphonate (ANS) to stain caseinate [32]. A stock solution of ANS (200 mg/L) was prepared in imidazole/acetate buffer and stored in dark at room temperature (25 ◦ C). ANS concentration was progressively increased until signal saturation. Cas concentration was kept constant at 0.33 g/L whereas EPS concentration ranged from 0 to 1 g/L. For each EPS concentration, 2.8 mL of complexes solution were mixed by vortexing with 5–40 ␮L of ANS solution. Fluorescence intensity emission was measured using a spectrofluorimeter (LS 55, Perkin Elmer, USA) after a scan of wavelengths made between 420 and 650 nm with an excitation wavelength of 380 nm. The scan speed was fixed at 500 nm/min. S0 was determined from the initial slope of the linear regression analysis of the maximal fluorescence intensity against ANS concentration (5–40 ␮L/2.8 mL).

could be derived from ATP by phosphorylation or from the sugar nucleotide. The change in the net charge of the different EPS samples after protein precipitation using TCA was studied by measuring zeta potential at pH values ranging from 2 to 9. It can be seen from Fig. 1 that before treatment with TCA, all EPS samples showed charge reversal from positive to negative when increasing the pH except for EPS-GM which has negative charge regardless of pH variation (Fig. 1D). After pretreatment with TCA, all EPS samples became negatively charged at the different studied pH values which indicate that the positive charge of EPS could be generated by the contaminating proteins where the anionic nature of the EPS samples is originating from phosphate groups [35]. Furthermore, it is important to note that EPS-GM was more negatively charged than the other tested EPS samples which was expected since it is the EPS that contains the highest phosphate content (1.02%) (Table 1).

2.5.7. Scanning electron microscopy (SEM) The microstructures of the formed EPS/Cas complexes were examined by scanning electron microscopy (Hitachi electron microscopy, TM 3030, Japan) at both pH 3.0 and pH 3.5, respectively. Complexes were fixed to a sample stub with double-sided sticky tape and visualized at 4000× magnification and an accelerating voltage of 15 kV.

3.2. Study of EPS/Cas interaction

2.5.8. Epifluorescence microscopy An optical microscopy (Axiovert 25 CFL, Prolabo, France) was used in the fluorescence mode to examine the complexes microstructures. Complexes suspensions were stained with 9 ␮L of Nile blue added to 1 mL of EPS/Cas solution to obtain approximately a dye-protein ratio of 1/100 (w/w). 5 ␮L of samples were observed by a 100× oil immersion objective lens. Digital image files were acquired with a Nikon F90X camera connected to a digital image processing system (AxioVision, Zeiss, Germany) [33]. 2.6. Statistical analysis All experiments were performed using at least three freshly prepared solutions and results were reported as the mean ± one standard deviation. Statistical analyses were performed using the IBM SPSS 19 statistics software. 3. Results and discussion 3.1. EPS characterization The composition analysis of EPS samples is reported in Table 1. As reported by Abid et al. [25], all the samples contain neutral monosaccharides and identified as mixtures of levan and dextran except for EPS-GM, which shows the presence of an additional Nacetyl-glucosamine. The molecular weights of all EPS samples are higher than 106 Da. The total sugar content in the different EPS samples is ranged between 89.4 and 95.6% and results obtained by ICP indicate that all the samples contain phosphate (ranged from 0.32 to 1.02%) (Table 1). As reported by Kumar et al. [34], phosphate

The study of EPS interactions with Cas was crucial to provide a better understanding of the behavior of these biopolymers when they are simultaneously present in dairy mixtures during processing and to predict their performance as functional ingredients. This interaction could be affected by various parameters such as the nature of biopolymers, polysaccharide/protein ratio as well as the pH value. 3.2.1. Influence of EPS to Cas ratio 3.2.1.1. Suspension turbidity of EPS/Cas complexes. Complexes formation was studied when different EPS concentrations (0–1 g/L) were added to Cas solution (0.33 g/L) at pH 3.0. By increasing EPS/Cas ratio (w/w), two phases can be distinguished (Fig. 2A): for EPS/Cas ratio lower than 1.8, turbidity was very low and remained almost constant. However, during the second phase (for ratios ranging from 1.8 to 3), higher turbidity was observed which reached its maximum at 1 g/L of EPS (corresponding to a ratio of 3) indicating thus an extensive biopolymer aggregation between positively charged Cas (at pH < pHi ) and negatively charged EPS (as shown in Fig. 1). In fact, an increase in turbidity can be indicative of an increasing of the biopolymer complexation between the two oppositely charged biopolymers driven by electrostatic interactions [36], and/or an increasing of the size of formed complexes that are large enough to scatter light [7]. Different results were obtained by previous works which studied lysozyme/low methoxyl pectin complexation and found that low concentrations of low methoxyl pectin (0.2 g/L) enabled maximal suspension turbidity, whereas higher concentrations resulted in a very slight aggregation of biopolymers indicating gradual dissociation of the aggregates to reform individual complexes [37]. Furthermore, it can be seen from Fig. 2A that EPS-GM generated the highest suspension turbidity compared to the other EPS samples and thus EPS-GM generated the highest biopolymers aggregation. This result was expected since EPS-GM is more negatively charged than the other EPS samples as it was concluded from Fig. 1. Hence, attractive interactions between EPS-GM/Cas are more susceptible to occur. It can be suggested that with small amounts of biopolymer, individual complexes were

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Fig. 1. Effect of pH on zeta potential of the different EPS solutions (1 g/L). (A): EPS-BMS; (B): EPS-TMS; (C): EPS-DPS; (D): EPS-GM. Discontinuous dot line denotes zeta potential values before TCA treatment; Continuous line denotes zeta potential values after TCA treatment.

formed and were slightly aggregated. Then, the increase of EPS concentration resulted in a maximal turbidity indicating extensive biopolymer aggregation as a result of individual complexes combination with each other through a bridging mechanism [37]. 3.2.1.2. Zeta potential of EPS/Cas complexes. The zeta potential (ZP) of the formed complexes was measured at pH 3.0 as a function of EPS/Cas ratio to examine how the net charge of the complexes changes (Fig. 2B). Although ZP of the different complexes decreased when increasing the EPS concentration, they remained positively charged probably due to an excess of positive charges deriving from Cas at these ratios. This finding could confirm that the involved interactions between EPS and Cas are electrostatic. Ye [36] reported that during complexes formation, there is gradual attachment of proteins on polysaccharide molecule resulting in a decrease in the overall net charge of the anionic polysaccharides. At EPS/Cas ratio equal to 2.9, only ZP of the complexes formed with EPS-GM and Cas achieved the electroneutrality (␨ = 0 mV). An excessive amount of anionic EPS in the medium resulted in decreasing ZP where greater amount is required to neutralize protein solution. At this charge neutralization point, the complexes exhibited high insolubility and stability since the highest interactions are established [5,38]. At EPS/Cas ratio of 3 (corresponding to 1 g/L of EPS), only EPS-GM showed charge reversal from positive to negative. Thus, it can be suggested that at pH 3.0, only EPS-GM had a net charge higher than that of Cas since it contained

the highest amount of phosphate compared to the other EPS samples (Table 1). Initially, anionic polysaccharides bind to the cationic proteins causing charge neutralization, leading thus to the formation of insoluble “protein-polysaccharide” complexes [39]. Further binding of anionic EPS on neutral protein aggregates makes it effectively anionic, leading to the formation of soluble and negatively charged complexes [40]. Similar findings were recently reported for complex coacervation between low methyl pectin and sodium caseinate [7].

3.2.1.3. Surface hydrophobicity of EPS/Cas complexes. As it can be seen from Fig. 2C, the surface hydrophobicity (S0 ) of free Cas (unbound to EPS) determined at pH 3.0, remained approximately constant when EPS concentration increased up to 1 g/L. This result indicates that the hydrophobic sites of Cas have not been masked even after the adsorption of anionic EPS. Whereas, in the case of EPS-GM, S0 significantly dereased when increasing EPS concentration (Fig. 2C). This result could be correlated to the presence of N-acetyl-glucosamine detected only in EPS-GM resulting in the hiding of free Cas hydrophobic sites. Consequently, further contribution of hydrophobic interactions in EPS-GM/Cas complex formation could be suggested [41]. This finding could explain the highest amount and/or size of the formed complexes with EPSGM (Fig. 2A). Doublier et al. [42] reported that non-electrostatic interactions, such as hydrogen bonding and hydrophobic interac-

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Fig. 2. (A): Effect of EPS to Cas ratio on the suspension turbidity of the solution at different EPS concentrations (0–1 g/L); (B): Effect of EPS to Cas ratio on the zeta potential of sodium caseinate solutions (0.33 g/L) in the presence of different concentrations of EPS (0–1 g/L); (C): Effect of EPS to Cas ratio on the surface hydrophobicity of sodium caseinate in the presence of different concentrations of EPS (0–1 g/L).

tions within the complex could lead to the formation of irreversible complexes. 3.2.1.4. Epifluorescence microscopic observation. The observation of microstructures of the formed complexes was carried out with an optical microscopy in the fluorescence mode at pH 3.0 with EPS concentrations ranging from 0 to 1 g/L. It can be noticed from results shown in Fig. 3 that the size of aggregates is proportional to the initial EPS concentration. Increasing initial EPS concentration resulted in higher complexation between oppositely charged biopolymers and combination between small individual complexes (formed at low EPS concentrations) with each other resulted into larger aggregates. The highest complex size was observed at EPS/Cas ratio of 3. These results are in accordance with the results related to turbidity and ZP measurements (Fig. 2A and B) and are also in agreement with those reported by Eghbal et al. [7] and Bayarri et al. [37] who studied pectin/Cas and lysozyme/pectin complexes, respectively. Moreover, it can be seen from Fig. 3 that complexes formed with EPS-GM were characterized by larger sizes compared to the other EPS samples, which is consistent with the results discussed above (Fig. 2). 3.2.2. Influence of pH 3.2.2.1. Suspension turbidity, granulometric size and microstructures of EPS/Cas complexes. The effect of pH on the complexes formation was investigated at both pH 3.0 and pH 3.5 with EPS and Cas concentrations fixed at 1 g/L and 0.33 g/L, respectively. pH is a key factor in the behavior of mixed biopolymer solutions due to its effect on the number of charges carried by the ionizable groups in the molecules. It can be seen from Fig. 4A that the highest absorbance

was observed at pH 3.5 for all the studied EPS samples. The pH effect supports electrostatic interactions between EPS and Cas. This increase in absorbance at 600 nm at pH 3.5 closer to the pHi of Cas (pH ∼ 4.60) is mainly due to the reduction in the electrostatic repulsion between complexed proteins as it was already reported [43] and the decrease of protein solubility [39]. In addition, this increase in turbidity can be due to electrostatic interactions between EPS (which are more negatively charged at pH 3.5 than at pH 3.0 as shown in Fig. 1) and the self-aggregated Cas (exposing positive patches on their surface). These facts may contribute to an increase of both size and number of the particles. This aggregation was also studied by particle size measurement at both pH 3.0 and pH 3.5, respectively (Fig. 4B). Results showed that aggregates formed at pH 3.5 were bigger than those formed at pH 3.0 which is in agreement with the obtained results related to turbidity. In fact, all the aggregates formed at pH 3.0, except for EPS-GM, were not large enough to measure their distribution sizes since these latter were lower than the detection limit of the instrument. However, at pH 3.5, biopolymers were more aggregated and had an average size (D [4,3]) ranging from 16.04 ␮m (EPS-DPS) to 46.51 ␮m (EPS-GM) which was confirmed by both epifluorescence microscopy and SEM at pH 3.0 and pH 3.5 (Fig. 4C and D, respectively). These results were expected since at pH 3.5, the net charge of EPS is higher (Fig. 1) and therfore the attractive electrostatic interactions between both of the biopolymers are more accentuated as it was shown in Fig. 4A.

3.2.2.2. Zeta potential of EPS/Cas complexes. As shown in Fig. 5A, ZP of EPS/Cas complexes decreased at pH 3.5 but remained positive either at pH 3.0 or pH 3.5. In contrast, ZP of the complexes

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Fig. 3. Microscopic structures of the formed EPS/Cas complexes stained with Nile blue as a function of EPS to Cas ratio.

Fig. 4. (A): Effect of pH on the turbidity of EPS/Cas solutions at constant concentrations of EPS (1 g/L) and sodium caseinate (0.33 g/L); (B): Effect of pH on the average size of the formed complexes (0.33 g/L of sodium caseinate and 1 g/L of EPS); (C): Epifluorescence microscopic observations of the formed complexes at both pH 3.0 and pH 3.5; (D): SEM observations of the formed complexes at both pH 3.0 and pH 3.5.

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Fig. 5. (A): Effect of pH on zeta potential of EPS/Cas solutions at constant concentrations of EPS (1 g/L) and sodium caseinate (0.33 g/L); (B): Effect of pH on the surface hydrophobicity of sodium caseinate in the presence of 1 g/L of EPS.

formed with EPS-GM remained negative at both pH values and its net charge at pH 3.5 was higher than that at pH 3.0. In fact, the negative charge densities of the different EPS are higher at pH 3.5 than at pH 3.0 (Fig. 1). Therefore, at pH 3.5, electrostatic interactions are more susceptible to occur with cationic sites of Cas and/or Cas aggregates and EPS can bind more molecules of Cas per molecule of EPS [44]. These results are in good accordance with those found and discussed above. As reported by Ye et al. [45], when the pH becomes closer to Cas pHi , proteins tend to aggregate in small scale prior to large-scale aggregation. Therefore, polysaccharides may be complexed to the small-scale aggregates via electrostatic interactions between the positive sites in the outside of protein aggregates and the negatively charged polysaccharides. It has been also suggested that the presence of these latter on the outside of protein aggregates could sterically stabilize them to prevent their aggregation [46]. Previous research was interested in the study of the interaction between ␤-lactoglobulin and sodium alginate in the pH range of 3–7. Results showed that depending on pH and the relative charge density of biopolymers, no interaction at pH 6 and 7, weak interaction at pH 5 and strong interactions at pH 3 and 4 were observed [44]. 3.2.2.3. Hydrophobicity of EPS/Cas complexes. Results presented in Fig. 5B showed that S0 of Cas decreased at pH 3.5 in comparison with pH 3.0. This result could be explained by two main reasons. The first one is that pH 3.5 is closer to pHi of Cas and thus the hydrophobic sites of Cas were hided because of the important Cas aggregation. The second reason is that at pH 3.5, EPS are more negatively charged than that at pH 3.0 and thus the hydrophobic sites of Cas were hided because of the EPS binding to Cas molecules. In addition, it can be noticed that S0 values obtained with EPS-GM were the lowest. This result was expected since it was concluded from Fig. 2C that in the case of EPS-GM, there is a possible contribution of hydrophobic interactions in complex formation. Although electrostatic interaction is the main force for complexation between polysaccharides and proteins, both hydrogen bonding and hydrophobic interactions play a secondary role for stability of the protein-polysaccharide aggregates [47]. Together with the results of the suspension turbidiy, it can be concluded that hydrophobic interactions are involved in the formation of EPSGM/Cas complexes. 4. Conclusion The present work aims to study the formation of complexes between oppositely charged sodium caseinate and different bacterial EPS samples as a function of polysaccharide to protein ratio

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