Influence of concentration and pH in caseinomacropeptide and carboxymethylcellulose interaction

Food Hydrocolloids 35 (2014) 170e180

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Influence of concentration and pH in caseinomacropeptide and carboxymethylcellulose interaction V.C.F. Burgardt a, *, D.F. Oliveira a, I.G. Evseev a, A.R. Coelho a, C.W.I. Haminiuk b, N. Waszczynskyj c a b c

Food Technology Department, Federal University of Technology e Paraná, P.O. Box 135, Francisco Beltrão, PR, Brazil Post-Graduation Program of Food Technology, Federal University of Technology e Paraná, P.O. Box 271, Campo Mourão, PR, Brazil Post-Graduation Program of Food Technology, Federal University of Paraná, P.O. Box 19011, Curitiba, PR, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 May 2012 Accepted 15 May 2013

Aqueous systems with caseinomacropeptide (CMP), with or without carboxymethylcellulose (CMC), at different concentration and pH (2, 4 and 6.5), were analyzed. Strong evidence of interaction between the peptide and the polysaccharide was found at all pHs, even those close to neutral. All samples formed gel at pH 2. The formation of soluble and insoluble complexes occurred at pH 4. FTIR technique with Fourier’s transformation was employed to analyze changes in the system. Results show visible changes in bands at different pHs and concentrations. Since amide I band had highest modification due to pH rather than to CMC, the interaction between CMC and CMP may be electrostatic. The rheological behavior was also changed and shifted from shear-thickening to shear-thinning in the presence of the polysaccharide. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Visual diagram FTIR Rheology Spectrophotometric measurements Interaction

1. Introduction Caseinomacropeptide (CMP) is a terminal peptide composed of 64 aminoacids caused by the cleavage of k-casein in the bonding Phe105-Met106, which occurs in the cheese fabrication process with rennet. CMP is released in the whey whereas the remaining kcasein, or para-k-casein, is precipitated in the mass (Delfour, Jolles, Alais, & Jolles, 1965). CMP has two genetic variants, A and B, and the posttranslational modifications glycosylation and phosphorylation. The peptide’s glycosylated part (gCMP) constitutes 50% of total CMP, with total carbohydrates present in k-casein. The predominant carbohydrate is N-acetylneuraminic acid (sialic acid) (Coolbear, Elgar, & Ayers, 1996; Mollé & Leonil, 2005). Its functional properties are highly interesting for the development of new health products. The component, with antithrombosis and immunomodulator effects, may be used in food for Phenylketonuria bearers (Chabance et al., 1995, 1998; Matin & Otani, 2000; Monnai & Otani, 1997; Otani, Horimoto, & Monnai, 1996; Otani & Monnai, 1995; Otani, Monnai, Kawasaki, Kawakami, & Tanimoto, 1995). Further, CMP is technologically characterized

* Corresponding author. Tel.: þ55 46 35237111; fax: þ55 46 35237017. E-mail addresses: [email protected], [email protected] (V.C.F. Burgardt). 0268-005X/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodhyd.2013.05.005

by emulsification and emulsion stabilization, the formation of gel and spray and others (Ahmed & Ramaswamy, 2003; Burton & Skudder, 1987; Chobert, Touati, Bertrandharb, Dalgalarrondo, & Nicolas, 1989; Marshall, 1991; Martín Diana, Frías, & Fontecha, 2005). Systems with proteins and polysaccharides are extensively used in the food industry. This is due to the fact that their interactions improve the proteins’ technological characteristics in the systems. Attraction or repulsion between proteins and polysaccharides may occur in solutions due to factors which include source, pH, ionic strength, temperature, concentration or shear (Cèsaro, Cuppo, Fabri, & Sussich, 1999; Delben & Stefancich, 1997; Dickinson, 1992). Through changes in pH or ionic strength of the aqueous phase, the proteinepolysaccharide interaction force passes from attraction to repulsion or vice-versa (Dickinson, 2008). The interaction between proteins and anionic polysaccharides, such as Carboxymethylcellulose (CMC), occurs more intensely with pH close to the isoelectric (pl) mark of the protein or of pKa of the polysaccharide. However, recent studies have shown that interaction exists even when pH is close to neutral, with the formation of soluble complexes (Benichou, Aserin, Lutz, & Garti, 2007; Koupantsis & Kiosseoglou, 2009; Tolstoguzov, 1997). CMC in biopolymer systems has a complexation capacity with whey proteins (Damianou & Kiosseoglou, 2006; Girard, Turgeon, & Paquin, 2002; Hansen, Hidalgo, & Gould, 1971; Kika, Korlos, &

V.C.F. Burgardt et al. / Food Hydrocolloids 35 (2014) 170e180

Kiosseoglou, 2007; Koupantsis & Kiosseoglou, 2009), and with a-lactalbumin and b-lactoglobulin (Capitani, Pérez, Pacheco, Teresa, & Pilosof, 2007). Although this fact shows the compatibility of the polysaccharide with different proteins from whey, investigations with CMP, which significantly contributes with other total proteins of the milk industry sub-product, are not found in the literature. Current work concentrates on the manner CMP interacts with CMC in different concentrations and at several pH conditions: close to neutral (6.5); peptide pl and CMC pKa (4) and sialic acid pKa (2). Infrared and spectrophotometry techniques were employed to verify whether there occurred a peptideepolysaccharide interaction and whether it was chemical or electrostatic. Changes in physical characteristics were investigated by visual diagram and rheology. 2. Materials and methods 2.1. Materials Assays employed (i) BioPURE-GMPÒ casein glycomacropeptide (CMP) provided by DAVISCO Foods International, Inc. (Le Sueur, MN, USA). CMP composition includes protein (dry base) 82.5% (m/m) (N  6.47), with CMP 90.0% (m/m) (N  7.07) of total protein, 0.5% (m/m) fat, 6.0% (m/m) ash and moist; (ii) carboxymethylcellulose (30 FGH e 70520, with high substitution degree and high viscosity) provided by International Specialty Products e Brazil. 2.2. Preparation of samples Samples were prepared in purified Milli-Q water from mixtures of stock solutions of caseinomacropeptide (CMP) and carboxymethylcellulose (CMC) to obtain the adequate concentrations. Further, pH 2, 4 and 6.5 were adjusted with HCl and NaOH (1 N). All experiments were in triplicate. 2.3. Visual diagram The different solutions with peptide (CMP) at concentrations 2, 4, 6, 8 and 10% (m/v), with the addition or not of polysaccharide (CMC) (0, 0.25 and 0.5% m/v) at pH 2, 4 and 6.5, stored at 25  C, were observed during 15 days. 2.4. Spectrophotometric measurements to evaluate CMPeCMC interaction Aqueous solutions of carboxymethylcellulose (CMC) and methylene blue (MB), respectively in concentrations 1% (m/v) and 0.002% (m/v), were mixed for a series of polysaccharide-dye solutions. Final MB concentration was 0.001% and CMC ranged between 0 and 0.5% (m/v). When the polysaccharide is added to the MB aqueous solution, the peak at 664 nm decreases and a shoulder at 615 nm are detected, that is enhanced with increasing the gum concentration. This absorption shift could be attributed to the absorption of MB molecules interacting with the gum (Michon, Konaté, Cuvelier, & Launay, 2002). In a second analysis, CMP was added to solutions with CMC and MB. So that the required final concentrations for CMP (0e8%), CMC (0.20%) and MB (0.001%) could be obtained, increasing CMP (0e16% m/v) levels were added to the solutions CMCeMB with 0.002% (m/ v) of MB and 0.4% (m/v) of CMC and pH 2, 4 and 6.5 were adjusted. Absorbance ratios 664 and 615 nm were reported due to CMP concentration. The interaction between the peptide and the polysaccharide triggered an increase in the ratio because the blue methylene molecules became free once more (Benichou et al.,

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2007). Three repetitions and 4 replicates were undertaken for each samples (n ¼ 12). 2.5. Infrared spectrometry Medium infrared spectra (4000e400 cm1) were undertaken to characterize CMP and CMC and to verify possible chemical interactions occurring at pH 2, 4 and 6.5 and in CMP and CMC concentrations of 4, 6 and 8% (m/v) and 0, 0.25 and 0.5% (m/v) respectively. Samples of mixture 150 mg of KBr (for IR spectroscopy, Merck Chemicals International) with 3 mg of each lyophilized sample were prepared. Material was placed in a formatting complex and the lozenges were formatted when an 8 ton/cm2 pressure by a hydraulic press was applied for 5 min. Spectra were obtained by Fourier Transformed Infrared Spectrophotometry (FTIR) BioRad model FTX 3500 (Hercules, CA, USA), with Silicon Carbide MIR source, Beam splitter MIR (extended KBr), detector MIR DTGS (deuterated triglycine sulfate), 3-Term BlackmaneHarris Apodization, in which 32 scans per sample were undertaken with 4 cm1 resolution. Spectra results were analyzed by Origin 8.0 (OriginLab Corporation, MA, USA). 2.6. Microstructure by confocal laser scanning microscopy (CLSM) Microstructure was analyzed by CLSM (Confocal Nikon Eclipse E800, Nikon Corporation Instruments Company, Japan) with 20X objective and laser Bio-Rad Radiance System e Argon 514 (Bio-Rad Microscience Ltd. 1999). Peptide was marked by adding a small quantity of concentrated solution of Rhodamine B in the solutions. A drop of each sample was then placed on the slide, covered by a slip and sealed to avoid evaporation. Incident light was emitted at 514 nm and fluorescence light ranged between 600 and 700 nm. Images were obtained by Laser Sharp 2000. All samples were prepared in triplicate. 2.7. Rheological behavior of solutions Since the interaction between proteins and anionic polysaccharides is a rather frequent event at a pH below the isoelectric point, only pH 6.5 samples were evaluated. Martinez, Farías, and Pilosof (2011) showed that dimers of CMP between pH 6.5 and 4.5 are negatively charged. Probably the interactivity within this pH with CMC became impossible and thus CMC influence in the rheological behavior of CMP solutions was investigated. Rheometer Brookfield DV III ultra (Brookfield Engineering Laboratories, Inc., Middleboro, MA, USA) with SC4-31 rotor was employed at sheering rates between 0 and 85 s1, at 25  C. Solutions were evaluated in triplicate and had different CMP (2e10%) and CMC (0e0.5%) proportions. Data adjustment model in the case of fluid polymers (Delben & Stefancich, 1997) was the power law (Equation (1)) in which all samples had a correlation coefficient close to 1 (>0.99). Statistical analysis of results was conducted by analysis of variance (ANOVA/ MANOVA) and mean Tukey test (p < 0.05) by Statistica 7.1 (StatSoft, Tulsa, OK, USA).

s ¼ k$g_ n where:

s ¼ shear stress (Pa)

k ¼ consistency coefficient (Pa sn)

g ¼ shear rate (s1)

n ¼ flow behavior index (dimensionless)

(1)

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3. Results and discussion 3.1. Visual diagram Samples remained in solution after a 15-day period in pH 6.5 and separation of phases did not occur, whereas assays formed gel when pH was 2. A White precipitate occurred at pH 4. However, the addition of CMC inhibited precipitation, excepting samples 5 and 8, with 4 and 6% CMP and the addition of 0.25% CMC, as Fig. 1 shows. It is highly interesting to note that when precipitate volume in samples 5 and 8 was compared with the neighboring tube containing the same CMP concentration and without CMC (samples 4 and 7), the polysaccharide was probably precipitating together with the peptide. In fact, an increase in precipitate volume occurred. The above empirical observation suggests the formation of coacervates (Fig. 1). Gel was formed in concentrations 8 and 10% CMP. In fact, pH 4 has a complex system, with several different events (complexation, precipitation and gelation) which may be explained by the peptide’s heterogeneity. Since commercial CMP is composed of aCMP and gCMP (non-glycosylated and glycosylated forms), different isoelectrical points occur (Mikkelsen et al., 2005). In the case of aCMP, a less acid part precipitates at pH 4.3e4.6, due to genetic variants of k-casein. However, total aCMP is not charged

only when pH varies between 4.0 and 4.1. In pH 4, gCMP isoforms with less sialic acid residues lies in the isoelectrical state, even though total gCMP reaches this state at pH 3.15 (Kreub, Strixner, & Kulozik, 2009). Therefore, only a part of the peptide’s total concentration was in an isoelectric state at pH 4. The above characteristic may explain gel formation in samples 10 and 13 (8 and 10% CMP, without CMC), in which gelification occurred even with precipitation (Fig. 1). Complexation produced soluble and non-soluble CMPeCMC complexes. This occurred because CMC is dissociated in pH 4 (pKa 4.4e4.2) (Keller, 1983). Free groups COO interact electrostatically with the positive charged peptide. Interaction causes the formation of electrostatic complexes with a subsequent increase in viscosity (Cluskey, Thomas, & Coulter, 1969; Keller, 1983). Since the formation of soluble complexes occurs with peptide isoforms above their pl (gCMP e glycosylated form). The interaction occurs because the peptide presents positive patches, when it is above pl and with a weak ionic strength (Weinbreck, Tromp, & de Kruif, 2004), due to the three lys residues (positively charged aminoacid) (Dziuba & Minkiewicz, 1996) and to N-terminal, which may react with the CMC carboxyl groups. As will be explained below, soluble complexes are also formed in pH 6.5. Since insoluble complexes or coacervates may be formed by proteins and polyelectrolytes, similar to CMC, in pH below the isoelectric point of proteins (Sacco, Bonneaux, & Dellacherie, 1988),

a

b

Fig. 1. Images of CMPeCMC mixtures at pH 4 in different concentrations after a 15-day period.

Fig. 2. Changes in Abs664/Abs615 rates for solutions with 0.001% Methylene Blue due to CMC concentration (a); or for mixtures 0.001% Methylene Blue e 0.20% CMC due to CMP concentration (b) at pH 2 (-); 4 (C) and 6.5 (:) at 25  C. Error bar: standard error (n ¼ 12).

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Fig. 3. FTIR spectrum for CMP.

Fig. 5. Comparative spectra for samples with the same CMP (4%) and CMC (0%) concentration at pH: 2 e, 4 e and 6.5 e.

insoluble complexes (tubes 5 and 8, Fig. 1), if in fact are extant, are formed with the non-glycosylated section of CMP with CMC. Coacervate systems have two visual phases, one of which is rich in polymers and the other in solvents. Polymers accumulate at the bottom of the tube (Bastos et al., 2010; Cooper, Dubin, Kayitmazer, & Turksen, 2005). Sample 8 shows clearly this behavior (Fig. 1).

This is because MB molecules are already bonded to the negatively charged regions and excess of polysaccharides does not interact (Benichou et al., 2007). Fig. 2b shows the influence of CMP addition with regard to Abs664/Abs615 in solutions with 0.001% MB added to 0.20% CMC. Absorbance rates increase to 664 nm since a displacement of the dye molecules is caused by peptide molecules. Since these molecules are released, a higher absorption occurs in the region. Fig. 2b shows that pH interfered in the CMPeCMC interaction, with a higher intensity at pH 2 and 4. However, interaction also

3.2. Spectrophotometric measurements According to Michon et al. (2002), there is an interaction between planar cation dye, such as methylene blue, and anion macromolecules (anionic polysaccharides) in diluted solutions. This interaction causes modification of the dye visible absorption spectrum. This is due to the formation of complexes among the components with dye electrostatic attraction for negative charged regions of polysaccharide. MB has a maximum absorption at 664 nm. Change in the spectrum occurs when the polysaccharide is added to the solution and maximum absorption occurs at 615 nm. Fig. 2a shows such change with regard to Abs664/Abs615, due to the addition of the polysaccharide (CMC) to the system. A decrease in the ratio Abs664/Abs615 occurs when 0.05% of CMC is added. Decrease is greater at concentration 0.1e0.2% of the polysaccharide. It may be perceived that from 0.25% CMC the ratio rates are greater than 1 and, therefore, absorbance at 664 is greater than at 615 nm.

Fig. 4. FTIR spectrum for CMC.

Fig. 6. FTIR spectrum for samples at pH 2.

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occurred when rates were close to neutral (6.5), which was corroborated by results obtained by other authors (Benichou et al., 2007; Koupantsis & Kiosseoglou, 2009; Michon et al., 2002). This fact is due to the formation of CMPeCMC soluble complexes, formed by aCMP or gCMP with CMC, since CMP is constituted by monomers (Martinez et al., 2011) in the latter pH. The formation of the above complexes is possible since the deprotonation of groups NH3 of Lys and N-terminal fails to occur up to pH 6.5. This fact warrants Hþ in positive dominions of CMP so that they could interact with the polysaccharide’s COO groups. According to Lesins and Ruckenstein (1988) and de Vries (2004), positive and negative dominions cause a non-uniform distribution of protein surface charges which benefits electrostatic interactions, even when the latter has the same charge as that of the adsorbent. Another hypothesis consists of the fact that Naþ ions act as counterions in the repulsion-reduced system between CMP and CMC. In the case of pH 2, interaction was higher than 4, this is due to the fact that dimers in the solution with a pH less than 3.15 are all positively charged (Martinez et al., 2011), which enhances electrostatic attraction by negatively charged carboxylic groups in CMC. On the other hand, a decrease of the ratio Abs664/Abs615 occurs in concentrations greater than 2% CMP. This fact suggests the beginning of phase separation in the sample, albeit at a microscopic scale. This is why it has not been observed in the visual diagram. Consequently, the lowest interactivity between CMP and CMC occurs. This may also be perceived at pH 4 in 8% concentration. Results that show phase separation were also found in the dynamic oscillation measurements (data not shown in current paper).

Fig. 7. FTIR spectrum for samples at pH 4.

3.3. FTIR 3.3.1. Characterization of CMP A probably overlying of bands occurs in the CMP spectrum between the 3400 and 3300 cm1 band (Fig. 3) in which the vibrational modes of the OeH stretching, free or H bonded, and bands related to secondary amides A (3300 cm1) and B (3080 cm1) are present. According to Twardowski and Anzenbacher (1994), secondary amides with TRANS have bands at 3300 and 3100 cm1, respectively corresponding to amide A and B. Amide A band corresponds to the stretching of NeH bond with the formation of hydrogen bond, whereas amide B results from FERMI resonance of the NeH stretching vibration with overtone of amide II vibration for TRANS. Bands are related to proline. Amide I and II lie respectively at 1648 and 1548 cm1. Amide l band is mainly attributed to C]O stretching of the peptide bond and is susceptible to different conformations of the secondary structure (Mangavel, Barbot, Popineau, & Guéguen, 2001). Amide II is characteristic of the combined movement of NeH vibrations and CeN stretching of the COeNH group (Bandekar, 1992; Lavialle, Adams, & Levin, 1982). Amide III band at 1240 cm1 represents the combination between vibration of CeN stretching and NeH bending of amide bonds and the vibration waggings of the CH2 groups of the backbone glycine and proline side-chains (Jackson, Choo, Watson, Halliday, & Mantsch, 1995). Band at 1320 cm1 is attributed to e COeN group (amine). Bands referring to asymmetrical and symmetrical vibration of the methyl group (CH3) are at 2970 and 2940 cm1 (Abraham,

Fig. 8. FTIR spectrum for samples at pH 6.5.

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Sajan, Joe, & Jayakumar, 2008) and deformation CH2 at 1450 cm1. The hydrophobic region on CMP accounts for the two bands. Axial deformation C]O occurs at 2100 cm1 and vibration of COO groups at 1400 cm1 (Nakai, Li-Chan, & Hirotsuka, 1994). The band corresponding to carbohydrate residues was obtained at 1075 cm1 (Barja, Lemos, & Toranzo, 1989; Stewart, 1965). A scantily perceived band lies at 990 cm1 from the phosphate groups (PO4) due to phosphorylated sites of peptide, (Ser 149 and 127) (Carmona & Rodriguez, 1986). Bands at 935 and 900 cm1 may be related to galactose monosaccharide, whereas a probable overlying of bands lies at 600 and 670 cm1. In fact, a wide band between 800 and 600 cm1 occurs as a result of the angular deformation outside the NeH wagging mode (Abraham et al., 2008). Further, an axial deformation of the CeS bond also occurs, probably due to methionine (Stuart, 1997). 3.3.2. Characterization of CMC Fig. 4 shows the bands for CMC. Band at 3425 cm1 refers to the OeH stretching in intramolecular/intermolecular hydrogen

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bonding (Tong, Xiao, & Lim, 2008), whereas CeH stretching occurs at 2930 cm1. The combination of stretching bands COO and OH deformation lies at 2130 cm1 and salt COONaþ is identified at 1602 cm1 (Zaleska, Tomasik, & Lii, 2002). Bands at 1420 and 1330 cm1 deal with C]O stretching in COO ions (Yuen, Choi, Phillips, & Ma, 2009), whereas CeO stretching in glycoside bond lies at 1270 cm1 and CeO stretching at 1160 cm1. Band at 1120 cm1 is due to CeC stretching. Bands 1060 cm1, which corresponds to stretching and deformation CeOH; 1020 cm1, which corresponds to OH deformation in glycoside bond; 900 cm1, which refers to CeH angular deformation, are found in the CMC digital impression. 3.3.3. Influence of pH and concentration of components in CMPe CMC interaction When identical formulations at the same CMP and CMC concentration from different pH rates are compared, the influence of this parameter in the components interaction is underscored. Change in the behavior and intensity of the bands with the

Fig. 9. Confocal laser scanning micrographs of CMPeCMC mixtures at pH 2.

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polysaccharide may be observed too. Mean and standard deviation of samples compared at different moments of the discussion were calculated. As a rule, the intensity of the spectra, with the same CMP and CMC concentration but with different pH (2, 4 and 6.5), is the same. However, when samples with 4% CMP concentration and without the addition of CMC are compared (Fig. 5), a more acute difference among the three pH may be observed. This is due to the aCMP precipitation in pH 4, as shown in the situation frame with a lower concentration of soluble peptide as a lower interaction among the monomers and in low intensity bands. The result confirms the observations of the visual diagram. However, when identical pH samples and peptide concentration are compared, with and without CMC addition, samples with carbohydrate (0.25 or 0.5%) have a higher intensity for all spectrum bands. The fact evidences CMPeCMC interaction. Results corroborate those obtained by spectrophotometry, which also show an interaction among biopolymers in the pH under analysis.

One narrow and low intensity band appears in pH 6.5 at 2038  0.5 (Fig. 6c). This band is not present in samples with other pH. This may be related to deprotonation and consequent formation of negatively charged CO groups in Asp and Glu lateral chains, with pH higher than 4 (Okuno, Iwase, Shinzawa-Itoh, Yoshikawa, & Kitagawa, 2003). The behavior of band at 1402  1 cm1 reinforces this fact since it is more marked in pH 6.5. This is due to the fact that the ionized form of the carboxyl groups provides a band at 1400e1420 cm1 and COOH at 1700e1750 cm1 (Nakai et al., 1994). Changes in amide I band at 1648 cm1 for CMP were more sharp at pH 2 (1637  1) (Fig. 6a) and 4 (1639  1) (Fig. 6b) than at 6.5 (1648  1) (Fig. 6c). This fact may be explained by a higher change in secondary structure of peptide and by a higher exposition of functional groups in acid pH. No significant changes occurred in the band, due to the polysaccharide, as Figs. 6e8 shows. The addition of the polysaccharide does not alter the absorption peak for the amide I band in the samples with the same CMP and pH concentration.

Fig. 10. Confocal laser scanning micrographs of CMPeCMC mixtures at pH 4.

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In other studies, the modification of amide I band and the occurrence of other new bands were attributed to the interaction between polymers through covalent links. Research by Gonzaga, Ricardo, Heatley, and Soares (2005) demonstrated that bands within the 1000e1171 cm1 interval indicated ester O-acetyl too, which made Xie et al. (2010) speculate on the presence of glucane protein complexes. Lii, Tomasik, Zaleska, Liaw, and Lai (2002) analyzed the interaction between CMC and gelatin in alkaline pH and noted a band which was not related to the spectra of these polymers, coupled to modifications in the amide I band. The formation of complexes by chemical bonds between the COO group of CMC and protein amide I could be thus described. According to results, it may be proposed that an interaction between components is purely electrostatic, since the amide I band was modified more sharply by pH rather than by the polysaccharide. Further, the bands of individual components were maintained in the spectra of the mixtures. The above results corroborate those by Guerrero, Retegi, Gabilondo, and de la Caba (2010) when they compared the region between 1200 and

177

800 cm1 in films of soybean protein with glycerol. No modifications in the characteristic peaks of the components and in the amide I band were reported in this case. The authors concluded that the interaction occurred without the formation of covalent bonds. The secondary trans amide band at 3281 1.80, 3282  1 and 3289  7 in pH 2, 4 and 6.5 (means  standard deviation of samples in each type of pH) underwent higher changes in pH close to neutral. This fact shows the interaction between the CMC’s COO groups and the peptide’s N-terminal, and reinforces the occurrence of interaction, without the deprotonation of NHþ 3 groups in this type of pH. Band of OeH bonds had the highest modifications in pH 2 and 4 (3405  3 and 3410  3) rather than in pH 6.5 (3409  2). 3.4. Microstructure Fig. 9 shows the samples microstructure at pH 2. Gel structure becomes more porous when CMC is added. Behavior is similar at pH 4 (Fig. 10) in samples with 8 and 10% CMP where gel formation occurred. In the case of systems with higher CMC, the intensification

Fig. 11. Confocal laser scanning micrographs of CMPeCMC mixtures at pH 6.5.

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of incompatibility between biopolymers is clearly noted. This results in phase separation and in the progressive increase of polysaccharides-rich phase extension (pores) (Olsson, Langton, & Hermansson, 2002). These results corroborate those obtained by spectrophotometric measurements which suggested phase separation or polymer incompatibility. Fig. 10 (pH 4), featuring concentrations between 4 and 6% CMP with CMC, shows the formation of spherical structures which contain polysaccharides and are surrounded by peptide. The above evidences the formation of coacervates in these samples. CMC addition clearly decreases the agglomeration of isoelectric CMP in samples with 2% peptide. It may be perceived that at pH 6.5 (Fig. 11) there is a homogenous distribution of the polysaccharide in CMC-less samples coupled to a slightly visible structure. Since, in the absence of CMC, the peptide organizes itself in small structures, dimers and monomers, a definite structure may not be perceived. The addition of polysaccharides causes the formation of a more particulated and rough structure. The above suggests structure strengthening when the polysaccharide is present. Consequently, an increase in samples viscosity occurs (see rheological data below).

solutions. On the other hand, shear-thinning behavior occurred when xanthan gum was added. Another study by Jambrak, Mason, Lelas, and Kresic (2010) reinforces the relationship between the decrease in molar mass and shear-thickening behavior in protein solutions, albeit with a-lactalbumin. Data corroborate those obtained in current study, since Martinez et al. (2011) suggest that the different CMP forms interact hydrophobically and constitute dimers (aCMPeaCMP, aCMPe

a

3.5. Rheological behavior Values of flow behavior index (n) (Table 1) were above 1 in solutions with only CMP and differed statistically among themselves only in the 2% solution. Rates were equal for the others (8, 6, 10 and 4%). CMP concentration did not only influence the rates for the parameter above but also the proportion of the added CMC, since the less polysaccharides added to the samples, the highest are n rates. Regardless of the concentration, shear-thickening behavior occurred in the solutions when only the peptide was present (Fig. 12a). On the other hand, in CMC-added samples, shearthinning behavior occurred, as the flow curves in Fig. 12b and c show. According to Alves (2002), the pseudoplastic fluids decreased the apparent viscosity when shear rate increased. This is due to hydrodynamic forces which become more intense and trigger a progressive rupture and lengthening of the system, with a resulting alignment with discharge and, consequently, a decrease in viscosity. Walkenström, Nielsen, Windhab, and Hermansson (1999) studied the influence of flow behavior in WPI aggregation in pure suspensions and in suspensions with xanthan gum. The authors perceived that changes in flux behavior enhanced the formation of small aggregates and the shear-thickening behavior in WPI

b

c

Table 1 Tukey test for parameters k (Pa sn) and n (dimensionless). CMP (%)

CMC (%)

K (Mean  standard deviation)a

n (Mean  standard deviation)a

2 2 2 4 4 4 6 6 6 8 8 8 10 10 10

0 0.25 0.50 0 0.25 0.50 0 0.25 0.50 0 0.25 0.50 0 0.25 0.50

0.015  0.004D 0.302  0.021D 2.563  0.188AB 0.011  0.001D 0.294  0.020D 2.968  0.187A 0.011  0.000D 0.247  0.005D 2.514  0.307AB 0.012  0.002D 0.252  0.042D 2.148  0.2781B 0.016  0.000D 0.337  0.047D 1.521  0.060C

1.150  0.073B 0.876  0.014CD 0.728  0.0159F 1.258  0.008A 0.889  0.007C 0.721  0.014F 1.306  0.005A 0.912  0.002C 0.745  0.024FE 1.322  0.037A 0.900  0.035C 0.775  0.025DEF 1.292  0.000A 0.870  0.028CD 0.847  0.003CDE

a Mean  standard deviation followed by same letters in the same column did not differ statistically according to Tukey test (p < 0.05).

Fig. 12. Flow curves for solutions with CMP (2% -, 4% C, 6% :, 8% ; e 10% =) (a), plus CMC (0.25%) (b) and CMC (0.50%) (c).

V.C.F. Burgardt et al. / Food Hydrocolloids 35 (2014) 170e180 Table 2 Tukey test for apparent viscosity (hap) (mPa s) at the shear rate of 50 s1 CMP (%)

CMC (%)

Apparent viscosity (mean  standard deviation)a

4 6 8 2 10 10 4 2 6 8 10 8 6 4 2

0.50 0.50 0.50 0.50 0.50 0.25 0.25 0.25 0.25 0.25 0 0 0 0 0

0.995  0.008A 0.923  0.025B 0.887  0.030B 0.882  0.010BC 0.836  0.024C 0.202  0.006D 0.190  0.008D 0.186  0.003D 0.175  0.002D 0.170  0.005D 0.049  0.000E 0.042  0.000E 0.036  0.001E 0.031  0.000E 0.026  0.000E

a Mean  standard deviation followed by same letters in the same column did not differ statistically according to Tukey test (p < 0.05).

gCMP, gCMPegCMP) which are negatively charged in pH ranging between 6.5 and 4.5. This fact shows that samples had a shearthickening behavior owing to the fact that the system was formed by densely packed small aggregates or dimers, confirmed by microstructure images. In their liquid phase, the aggregates are spread and form empty spaces when submitted to shear stress. The latter impairs the capacity of the solvent in lubricating and filling the interstitial spaces. Consequently, surface tension forces arise which make the system more solid (Glicksman, 1982). In contrast to what occurred in the case of parameter n, the samples with only CMP in the consistency coefficient (k) had the lowest rates, preceded by those with 0.250 and 0.50% CMC. Values of apparent viscosity (hap) at the shear rate of 50 s1 were evaluated for all samples. Table 2 indicates that samples with the highest rates are those with an addition of 0.50% of CMC, followed by those with 0.25%; finally those with 0%. Results demonstrate a positive interaction between CMPeCMC and confirm results obtained by other analyses which indicate the formation of soluble complexes in pH 6.5. 4. Conclusions FTIR results show that the interaction between CMC and CMP is electrostatic. All samples at pH 2 formed gel and the incompatibility of polymers was evident. At pH 4 peptide precipitation occurred and CMPeCMC complexes were formed. The latter also occurred at pH 6.5. The rheological behavior shifted from shear-thickening to shear-thinning with polysaccharides, albeit without any phase separation at this pH. Acknowledgments Current research was funded by the Federal University of Technology e Paraná and the Federal University of Paraná, Brazil. We would like to thank researcher Lucimara A. Forato of the Institute of Chemistry of São Carlos e University of Sao Paulo, Brazil. References Abraham, J. P., Sajan, D., Joe, I. H., & Jayakumar, V. S. (2008). Molecular structure, spectroscopic studies and first-order molecular hyperpolarizabilities of pamino acetanilide. Spectrochimica Acta Part A e Molecular and Biomolecular Spectroscopy, 71, 355e367. Ahmed, J., & Ramaswamy, H. S. (2003). Effect of high-hydrostatic pressure and temperature on rheological characteristics of glycomacropeptide. Journal of Dairy Science, 86, 1535e1540.

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