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Soluble protein fractions from pH and heat treated sodium caseinate physicochemical and functional p


Food Research International 33 (2000) 637±647

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Soluble protein fractions from pH and heat treated sodium caseinate: physicochemical and fu

nctional properties
Firouz Jahaniaval a, Yukio Kakuda a, Varghese Abraham b, Massimo F. Marcone a,*
a

Department of Food Science, University of Guelph, Guelph, ON, N1G 2W1, Canada b Caravelle Foods, Brampton, ON, L6T 5H4, Canada Received 11 November 1999; accepted 7 March 2000

Abstract The physicochemical (solubility and hydrophobicity), and functional (emulsifying activity index and emulsifying capacity) properties of soluble sodium caseinate fractions were studied as a function of pH (3±8) and temperature (50±100 C). Solubility was determined by measuring protein with the Bradford and 280 nm absorbency methods. Hydrophobicity was determined ?uorometrically with 1-anilino-8-naphtalenesulfonate (ANS), and cis-parinaric acid (CPA). Sodium caseinate solubility was minimal at pH 3.75±4 but the ANS and CPA-hydrophobicities and the functional properties of the soluble proteins increased in this pH range. Circular dichroic and 280 nm absorptivity measurements detected conformational changes. SDS-PAGE and reversed phase HPLC revealed substantial losses of as1 and b caseins following pH and heat treatment (pH 3.75 and 92.5 C) and the concomitant appearance of modi?ed compounds. Under these same conditions, the o-phtaldialdehyde values increased suggesting partial hydrolysis of sodium caseinate. The soluble protein fractions from sodium caseinate heat treated near the pI of the caseins were shown to have enhanced emulsifying activity and capacity. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Sodium caseinate; Soluble protein fraction; Physicochemical properties; Isoelectric point; Heat treatment; Functional properties; Hydrolysis

1. Introduction Sodium caseinate is a valued food ingredient due to its functional properties of emulsi?cation, water binding, fat binding and texturization (Kinsella, 1984; Southward, 1989). The major caseinate constituents are as1-, as2-, b-, and k-casein, in the proportion of 3:0.8:3:1 by weight (Aoki, Uehara & Yonemasu, 1996). Proteins as1-casein and b-casein, in roughly equal proportions, make up $75% of total bovine milk casein (Casanova & Dickinson, 1998). Among those proteins, b-casein has the greatest surface-active properties (Mulvhill & Fox, 1989), and is slightly more hydrophobic and surfaceactive than as1-casein (Dickinson, Rolfe & Dalgleish, 1988). Sodium caseinate has been shown to be remarkably heat-stable at pH=6.5 (Guo, Fox, Flynn & Mahammad, 1989) and highly insoluble at the isoelectric

* Corresponding author. Tel.: +1-519-824-4120; fax: +1-519-8246631. E-mail address: mmaarcone@foodsci.uoguelph.ca (M.F. Marcone).

point (pI) (Lieske & Konrad, 1994). The physicochemical and functional properties of sodium caseinate, depend on molecular ?exibility (Swaisgood, 1993) and extrinsic factors such as pH and temperature (Lee, Morr & Ha, 1992; Lieske & Konrad, 1994), ionic strength (Casanova & Dickinson, 1998), and the modi?cation of functional groups (Van Hekken, Strange & Lu, 1996). The molecular ?exibility of sodium caseinate is re?ected by its susceptibility to limited proteolysis, which dramatically changes functionality (Swaisgood, 1993). It has been shown that heating sodium caseinate solutions (2.5% pH 1±11) at 140 C for 120 min produces varying amount of pH 4.6 or 2% TCA-soluble peptides. The extent of hydrolysis decreased with increasing pH to a minimum at pH 6-7 (Hustinx, Singh & Fox, 1997). Physicochemical and functional properties of casein and sodium caseinate have been studied by many investigators (Guo, Fox, Flynn & Kindstedt, 1996; Guo et al., 1989; Hustinx et al., 1997; Konstance & Strange, 1991; Lee et al., 1992; Lieske & Konrad, 1994; Morr, 1985; Mulvhill et al., 1989; Pearce & Kinsella, 1978;

0963-9969/00/$-see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0963-9969(00)00108-3

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Singh, 1993), however, most studies have not focused on the pI region of the caseins. The objectives of this study were to determine the properties of the proteins remaining in solution after heat treatment near the pI range (pH 3.5±4.0) of caseins, and to compare them to the properties of the soluble proteins collected outside the pI range. 2. Materials and methods Sodium caseinate (Alanate 180, Lot 13C1804874R0003) with the following composition: 92.7% protein (N x 6.38)%, 3.5% ash, 4.2% moisture, 0.8% fat, 0.1% lactose, and < 0.01% antibiotics (IU/g), was obtained from New Zealand Milk Products Inc. (Santa Rosa, CA). as- b- and k-casein, ANS (1-anilino-8-naphtalenesulfonate), CPA (cis-parinaric acid), and o-phtaldialdehyde (OPA), were purchased from Sigma Aldrich Canada Ltd. (Oakville, ON). Canola oil (bleached and deodorized) was purchased from Dominco Inc. (Toronto, ON.). The OPA solution (25 ml of 100 mM sodium tetraborate, 2.5 ml of 20% w/w SDS; 1 ml of 40 mg/ml OPA in methanol, and 100 ml of b-mercaptoethanol), was prepared fresh and diluted to 50 ml with distilled and deionized water as described by Church, Swaisgood, Porter and Catignani (1983). Twenty percent homogeneous Phastgel was purchased from Pharmacia Biotech (Baie d'Urfe, Quebec, Canada). Bovine serum albumen and the Bradford reagent were purchased from Bio-Rad Co. (Richmond, CA). 2.1. Heat treatment For solubility, hydrophobicity, functionality and CD spectroscopy, 5 g of sodium caseinate was weighed into a 1000-ml beaker and dissolved in distilled and de-ionized water (approx. 700 ml at 25 C) using a magnetic stirrer. The solution was transferred to a 1000-ml volumetric ?ask, brought to volume and thoroughly mixed. Fifty milliliters (0.5% w/v) were placed into a 100 ml beaker and adjusted to speci?c pHs (3±8) using 1 N HCl or 1 N NaOH (Chung & Ferrier, 1992). The solutions were then heat treated in a water bath (VWR Scienti?c Co. Model 1160A) for 5 min (excluded come up time) at speci?ed temperatures (50±100 C). For the RP-HPLC and OPA assays, the initial concentration of sodium caseinate was 5% instead of 0.5%. The pH-temperature combinations were based on a central composite design (see statistical analysis section). 2.2. Protein solubility The solubility of sodium caseinate was determined by the Bradford (Bradford, 1976) and 280 nm absorbency

(Konstance & Strange, 1991) methods. The pH adjusted protein samples (0.5% w/v) after heat treatment were cooled to 25 C and centrifuged for 30 min at 25,000 g (J2-MC centrifuge, Beckman Instrument Inc., Palo Alto, CA), to sediment insoluble proteins. The supernatant was diluted 1:10 with pH adjusted distilled and de-ionized water and assayed for protein by the two methods. The 280 nm absorbency values were determined with a UV-260 spectrophotometer, (Shimadzu, Tokyo, Japan). A BSA standard curve (Bradford assay) and the% protein solubility was computed by the method of Lee et al. (1992) and Morr (1985) (%) PS=(protein in supernatant/total protein) ? 100. 2.3. Hydrophobicity Protein hydrophobicity was determined by the method of Kato and Nakai (1980). The 0.5% (w/v) protein solutions were prepared as described in the protein solubility section. The supernatants were diluted with pH adjusted distilled and de-ionized water to make a series of protein solutions ranging from 0.002 to 0.1%. Ten microliters of CPA (3.6 x 10?3 M in absolute ethanol containing 10 mg/ml of BHA) were added to a 2-ml aliquot of each diluted solution. The relative ?uorescence intensities of the CPA-protein conjugate were measured with a spectro?uorometer (RF-540, Shimadzu, Tokyo, Japan). The excitation and emission wavelengths were 355 and 415 nm, respectively. The same procedure was used to prepare protein samples for the ANS probe (aromatic hydrophobicity). Ten microliters of ANS (0.8 mM in 0.1 M phosphate bu?er pH 5.5) were added to a 2 ml aliquot of each diluted solution. The excitation and emission wavelengths were 380 and 475 nm respectively. 2.4. OPA assay The method of Church et al. (1983), was used for the OPA assay. Sodium caseinate (5%) solution was adjusted separately to pH 3.0, 3.75, 5.5, 8.0 and 6.78 (control) and heat-treated at 92.5 C for 5 min as described previously. The soluble fraction at pH 3, 5.5, 8 and control was diluted to 1: 100 with pH adjusted distilled and deionized water for protein assay and 1:20 for OPA assay. At pH 3.75 the dilution for protein was 1:5 and for the OPA assay, no dilution was required. Aliquots (100 ml) were pipetted directly into 3 ml cuvettes containing 2 ml of OPA reagent, mixed brie?y by inversion and incubated for 2 min at room temperature. The absorbency of the reaction mixtures was measured at 340 nm. In a second experiment, 100 ml of the supernatant from heat treated samples (5% sodium caseinate adjusted only to pH 3.75 and heat treated at 92.5 C for 0, 10

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and 20 min) were pipetted directly into a 3 ml cuvette containing 2 ml of OPA reagent and assayed as described above. 2.5. Gel electrophoresis (SDS-PAGE) The SDS-PAGE was carried out on sodium caseinate and protein soluble fractions after pH and heat-treatment (pH=3.75 at 92.5 C for 0, 10 and 20 min). The protein soluble fractions were freeze dried and dissolved in SDS solution (2% protein). Aliquots (approx. 1 ml) of treated samples were loaded onto a 20% homogeneous Phastgel (Pharmacia LKB-Phast system) and run according to the method of Dalgleish and Banks (1991) with slightly longer separation time. The protein bands were stained using Coomassie blue, and the stained gels were scanned at 633 nm using an Ultrascan densitometer (Pharmacia LKB). 2.6. RP-HPLC The reversed phase HPLC method of Bars and Gripon (1993) with slight modi?cations was used to determine the elution pro?le of the heat-treated caseinates. Samples (5% w/v sodium caseinate) were adjusted to pH 3.75 and heat treated at 92.5 C for 0, 10 and 20 min. The samples were cooled to 25 C and the insoluble proteins removed by centrifugation at 25,000 g. A nonheat-treated sample at pH 6.78 was used as the control. A gradient system (Waters 600 E, Millipore, Milford, MA) ?tted with a Econosil C18, 5 mm, 4.6 ? 250 mm column (Alltech, Deer?eld, IL) was used. The solvent system consisted of 0.115% (v/v) tri?uoroacetic acid (TFA) in water (solvent A), and 0.10% (v/v) TFA in 60% acetonitrile (solvent B). A linear gradient from 45 to 90% solvent (B) in 70 min was used to elute the peptides and proteins. Thirty microliter aliquots of the supernatant were automatically injected (Waters 700 Satellite WISP, Millipore, Milford, Mass.) and eluted at a ?ow rate of 1 ml/min. The e?uent was monitored with a Waters 486 UV detector at 214 nm. 2.7. Far-UV circular dichroism spectroscopy Circular dichroic measurements were carried out in the far-UV (190±250 nm) at 20 C under constant nitrogen purge using a Jasco J-600 spectropolarimeter (Japan Spectroscopic Co. Ltd., Tokyo, Japan) with a cell length of 1 mm. The supernatants from three samples (0.5% w/v sodium caseinate adjusted to pH 3.0, 3.75 and 8.0 and heat-treated at 100 C for 5 min) and one control (pH 6.78, no heat treatment) were diluted to a ?nal concentration of 0.1±0.2 mg/ml in distilled water. The% secondary structure was determined using the Jasco SSE program, which is based on the algorithm of Chang, wV and Yang (1978) and the database of Hennesseg and

Johnson (1981). Analyses were performed in triplicate with four scans per replicate. 2.8. Emulsifying activity index Emulsifying activity index (EAI) was determined by the turbidimetric method of Pearce and Kinsella (1978) with slight modi?cations. The 0.5% (w/v) sodium caseinate solutions were prepared as described in the protein solubility section. Six milliliters of supernatant and 2 ml liquid canola oil were placed into a polyethylene tube (11HH ? 4HH ) and homogenized with a Tekmar Tissumizer (Cincinnati, OH) at 20,000 rpm for 30 s (Lee et al., 1992). Immediately after homogenization, 100 ml of each emulsion was diluted 1:100 (v/v) with pH adjusted distilled and deionized water and the absorbency read at 500 nm. Calculations were performed according to the following equations: T ? 2X303 AaL ??e?r?e 8 uinsell?Y 1978? ?I?

where, T is the turbidity, A is absorbency at 500 nm, and L is the cell length (meters) and ? ? EAI ? 2Ta9C m2 ag ???tel 8 uil?r?Y 1990? ?P?

where, j is the volume fraction of oil phase (%) and C is the protein concentration in unit volume (g/m3) of protein in the aqueous phase. 2.9. Emulsifying capacity The method of Hung and Zayas (1991), with slight modi?cations was used to measure the emulsifying capacity (EC). The 0.5% (w/v) sodium caseinate solutions were heat treated and centrifuged as described in the protein solubility section. Ten milliliters of each supernatant were pipetted into plastic tubes (1HH ? 4HH ) and homogenized with a Tekmar Tissumizer at 20,000 rpm for 1 min, as canola oil was added to the tube at a ?ow rate of approximately 20 ml/min. During emulsi?cation, the conductivity was monitored with a conductivity meter (Hanna Instruments, Singapore). In an oil-in-water emulsion, the aqueous phase is conductive, but the conductivity will drop if a phase transition occurs or the emulsion system breaks down. The electrical conductivity was measured during the addition of oil and the total volume of canola oil was recorded at the point where the emulsion broke. The EC was calculated with the following equation: EC ? 9XaC ?g oilag protein? ?Q?

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where 9 is the volume of oil emulsi?ed by protein solution,  is the density of oil, and C is the grams of protein in the aqueous phase. 2.10. Statistical analysis Response surface methodology and the central composite design from Khuri and Cornel (1996) were used. The design with ?ve levels of temperature (50, 57.5, 75, 92.5 and 100 C), and ?ve levels of pH (3, 3.75, 5.5, 7.25 and 8) required 13 experiments. The heating time was 5 min for all treatment combinations. A second order equation and the PROC GLM and PROC CORR from SAS statistical software (SAS Institute, 1985, Cary, NC) were used for data analysis. The Sigma Plot software was used to plot three-dimensional response surfaces. All experiments were replicated twice, with two observations taken per analysis. 3. Results and discussion 3.1. Solubility Protein solubility can be expressed as nitrogen solubility index (Morr, 1985), water-soluble nitrogen, watersoluble protein or protein dispersibility index (Morr; Wolf & Cowan, 1975). In this study the solubility of sodium caseinate was determined by assaying for protein using the Bradford and 280 nm absorbency methods. Solubility was shown to be independent of temperature, but signi?cantly related to pH (P<0.020 and P<0.0034, for Bradford and 280 nm respectively). This was expected, since sodium caseinate has a random structure and is extremely heat-stable at pH 56.5 (Guo et al., 1989). The solubility dramatically increased as the pH increased from 6.5 to 8 (Fig. 1a) which is in close agreement with the solubilities reported by Lee et al. (1992) for the same pH range. The Bradford method places the minimum solubility around pH 3.5 to 4, which is near the isoelectric points of the casein molecules. Between pH 6.5 and 8, sodium caseinate exists as a polydispersed mixture of four major casein molecules, as1-CN, as2-CN, b-CN and k-CN. The molecular weights of these proteins vary from 19,000 (k-casein); 22,000±25,000 (as1-casein and as2-casein); and 24,000 (b-casein) Dalton (Eigle et al., 1984; Lee et al., 1992). The 280 nm absorbency readings produced the same solubility patterns between pH 6.5 and 8 as seen with the Bradford assay (Fig. 1b). However, at pH values 3.75±3.0, the 280 nm absorbency readings increased more rapidly than the Bradford solubilities. The large increase in absorbency appeared to be caused by an increase in absorptivity and not solely to an increase in solubility (Fig. 1b). Support for this is given in Table 1. Sodium caseinate (0.5% w/v) was adjusted to pH 3.0.

Fig. 1. Solubility of sodium caseinate as a function of pH and heat treatment (a) Bradford method, (b) absorbency at 280 nm.

Table 1 Protein concentration and 280 absorbency of the soluble protein fractions (initial protein concentration 0.5%) pH 3.00 3.75 5.5 7.25 8.00 Temp. ( C) 75 75 75 75 75 Proteina (mg/ml) 286?18 68? 5.57 122?7.04 401?15.06 414?2.92 280 nm (absorb.)a 0.590?0.000 0.217?0.053 0.113?0.010 0.321?0.002 0.324?0.003

a Supernatant diluted 1:10 with distilled water and analyzed for protein by Bradford.

3.75, 5.5, 8.0 and control and heat-treated for 5 min at 75 C. The treated samples were centrifuged and ?ltered through 0.2-mm cellulose acetate ?lter and the protein concentration determined by the Bradford method. The

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lowest protein concentration (68 mg/ml) occurred at pH 3.75 and represents a solubility of only 14.5%. This was expected due to the low solubility of caseins near their isoelectric points. However, the 280 nm absorbency for this sample was 0.217, which is 3±4 times greater than the other samples when calculated on a per mg protein basis. This increase in absorptivity may be related to conformational changes in the proteins at the lower pH and changes in composition. Although the caseinates have the lowest solubility at their pI, the wide range of pIs from 3.8 to 5.8 (Eigle et al., 1984) for the individual caseins, can result in a precipitate with varying casein compositions and as a consequence a soluble casein fraction that varies in composition. The amount and composition of the soluble proteins would depend on the pH and heat treatment given to the sample as well as the protein type, protein conformation, and the distribution of hydrophobic and hydrophilic amino acid residues on the surface. The solubility of caseins and the e?ect of pH and temperature on their interactions have been studied by Muller and Hayes (1963), Fox et al. (1982), Konstance and Strange (1991) and Lee et al. (1992). Our results show that near the pI (pH 3.75±4) where the caseins have minimum solubility, the soluble protein fraction exhibited greater hydrophobicity (Fig. 2a and b) and enhanced functional properties when compared to higher pHs. 3.2. Hydrophobicity Changes in aromatic and aliphatic hydrophobicities as a function of pH and heat treatment are shown in Fig. 2a and b. The ANS-hydrophobicity (Fig. 2a) was signi?cantly related to pH*temperature interaction (P<0.041). The hydrophobicity values increased with decreasing pH and increasing heat treatment (75± 92.5 C). CPA hydrophobicity (Fig. 2b) was signi?cantly related to pH*pH (P<0.043) and pH* temperature (P<0.035) interactions. The CPA-hydrophobicity increased with increasing heat treatment and decreasing pH. The data indicate that maximum ANS and CPAhydrophobicity occurred around pH 3±3.75. Similar increases in CPA hydrophobicity have been observed near the pIs of whey protein isolates (unpublished results) and globular protein from Amarantus hypochondrincus (Marcone & Yada, 1992). This increase in hydrophobicity may be due to a change in protein composition or modi?ed proteins that are soluble near their pI and have conformations in which more aromatic and aliphatic amino acids residues are exposed to the surface. The quantum yield of the ANS probe has been documented to be insensitive to pH in the range 2± 8 (Gibrat & Grignon, 1982). Therefore, it appears that the e?ects of pH on ANS hydrophobicity are due mainly to protein±probe interaction.

Fig. 2. Hydrophobicity of the soluble protein fractions as a function of pH and heat treatment; (a) ANS hydrophobicity, (b) CPA hydrophobicity.

3.3. o-Phtaldialdehyde assay The possibility that mild protein hydrolysis occurred at pH 3.75 was investigated. Three separate samples of sodium caseinate (5% w/v) were prepared and adjusted to pH 3.75. The samples containing both soluble and insoluble proteins were heat treated at 92.5 C for 0, 10 and 20 min and centrifuged. Aliquots (soluble fraction) were taken and analyzed for N-terminal amino groups by OPA and for protein by the Bradford method as described in method section. The results are shown in Table 2. As the heating time increased, the Bradford and the OPA values of the supernatants increased. A possible explanation for the increase in both the OPA and Bradford values could be the partial hydrolysis of the soluble and insoluble sodium caseinates when heattreated near their isoelectric points. Mild hydrolysis of

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the soluble caseins would increase the n-terminal value while hydrolysis of the insoluble caseins would release more soluble peptides and increase both the protein concentration and n-terminal groups. To show that the increase in the OPA values at pH 3.75 was due mainly to protein hydrolysis, the following experiment was performed. Sodium caseinate samples (5% w/v) were adjusted to pH 3, 3.75, 5.5, 8 and control (pH 6.78), and heated to 92.5 C for 5 min and centrifuged. The supernatant at pH 3.75 had the lowest protein concentration (1.1 mg/ml), but the highest OPA value of all the samples (Table 3). Although the protein concentration at pH 8, 5.5 and 3 were higher than at pH 3.75, the concentration of free amino groups in those samples was low. The implication is that the protein concentration does increase the OPA values, but not to the extent seen in the samples at pH 3.75. The data from Tables 2 and 3 support the possibility of mild protein hydrolysis near the pI of sodium caseinate. The hydrolysis of sodium caseinate over the pH range of 1±11 has been reported by Hustinx et al. (1997). The limited proteolysis of casein by plasmin has been shown to expose di?erent domains from b-casein (residues 29209; 106-209; 108-209), which are hydrophobic in nature (Swaisgood, 1993). Caessens, Gruppen, Visser, van Aken and Voragen (1997) showed that the hydrophobic peptide fractions produced from b-casein by plasmin hydrolysis (C-terminal half of b-casein sequence), possessed improved foaming and stabilizing properties compared to those of intact b-casein, especially at pH 4.0. They also showed that all peptide fractions had enhanced foaming properties as compared to intact bcasein, but SUP-4 (hydrophobic peptide) formed a stable emulsion at pH 4.0. The e?ects of partial hydrolysis of ovalbumin at pH 3.0 was reported by Honma et
Table 2 OPA analysis of the soluble protein fractions after heat treatment at pH 3.75 and 92.5 C (initial protein concentration 5%) pH 3.75 3.75 3.75 Temp. ( C) 92.5 92.5 92.5 Heating time (min) 0 10 20 Protein concentration (mg/ml) 0.840?0.01 1.74?0.04 2.52?0.06 OPA absorb. at 340 nm 0.473?0.008 0.585?0.021 1.301?0.014

al. (1991) while Matsudomi et al. (1985) showed that mild acid hydrolysis (0.05N HCl, 95 C for 30 min) increase surface hydrophobicity and greatly improve the solubility, emulsifying and foaming properties of soy proteins. In their study the increase in functional properties of soy proteins was attributed to deamination of asparagine and glutamine and not to cleavage of peptide bonds. 3.4. SDS-PAGE The composition of the soluble protein fractions from treated sodium caseinate (pH 3.75 and 92.5 C for 0, 10 and 20 min), was determined by SDS-PAGE and the results are shown in Fig. 3. Lane 1 (control) shows the typical electrophoregram of sodium caseinate with three major casein bands (k, b, and a-casein with percent areas of 17.5, 34.2 and 48.7%, respectively). The total protein and in particular a-casein were reduced when the pH was adjusted to 3.75. The two major bands remaining in the supernatant were k and b-caseins (lane 2). Their respective percentage areas were 53.6 and 46.4%. The increase in the k/b-casein area ratio (1.15:1) at pH 3.75 compared to the control (0.51:1) was due partly to the selective precipitation of b-casein but also to an increase in soluble proteins having the same

Fig. 3. SDS-PAGE of the soluble protein fraction from heat-treated samples. Sodium caseinate (lane 1, control); pH=3.75, no heat treatment (lane 2); pH=3.75, at 92.5 C for 10 min (lane 3); pH=3.75, at 92.5 C for 20 min (lane 4). Standard a s-CN, b-CN and k-CN (lanes 5, 6 and 7, respectively).

Table 3 OPA analysis of the soluble protein fractions (initial protein concentration 5%) after heat treatment at 92.5 C for 5 min pH Control 8.0 5.5 3.75 3.0 Temp. ( C) 92.5 92.5 92.5 92.5 92.5 Heating time (min) 5 5 5 5 5 Protein concentration (mg/ml) 35.7 32.3 25.8 1.10 26.0 Protein concentration used for OPA (mg/ml) 1.785 1.615 1.290 1.10 1.30 OPA absorb. at 340 nm 0.376 0.367 0.341 0.415 0.315 OPA/protein (absorb./mg) 0.210 0.227 0.264 0.372 0.242

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migration rate as k-casein. This e?ect was more pronounce in the heat-treated samples (92.5 C for 10 and 20 min), where the bands corresponding to k-casein are clearly increasing in concentration (Fig. 3, lanes 3 and 4). After heat-treatment a-casein was no longer visible and b-casein was only faintly visible. The majority of the a- and b-casein were precipitated when the pH was adjusted to 3.75, but the subsequent heat-treatment appears to have further altered the composition and properties of the soluble fraction. These changes were made evident by the increasing concentration of the area corresponding to k-casein and the production of lower molecular weight proteins or peptides that appeared as faint bands in the upper part of the gel. These compounds may represents some of the hydrolyze products detected by the OPA assay. 3.5. RP-HPLC The HPLC chromatograms of the supernatant from sodium caseinate at pH 3.75 (92.5 C for 0, 10 and 20 min.) and pH 6.78 (unheated control) are shown in Fig. 4. Two major peaks (as1 and b casein) are clearly seen in the native sodium caseinate (unheated control) with area percentages of 28.6 and 45.4% and retention times of 30.0 and 39.8 min, respectively; while k-casein appears as a broad peak (retention time 10±25 min) with area percentage of 26% (Fig. 4a). After adjustment to pH 3.75 (no heat-treatment), the area count for k and b casein remaining in the supernatant were 29.4 ? 106 and 33.6 ? 106 mV-s, respectively. The as1-casein was completely removed from the supernatant (Fig. 4b). However, incubating at 92.5 C for 10 and 20 min resulted in a substantial increase in the area count for material eluting with retention times between 10 and 30 min. Three broad peaks with retention times of 17.9 and 20.9 min (total area 37.9 ? 106 mV-s) and 41.5 min (area 34.0 ? 106 mV-s) were dominate features of the elution pro?le after 10 min of incubation (Fig. 4c). After 20 min of incubation, there was a further increase in area (46.7 ? 106 mV-s) for material eluting with retention times between 10 and 30 min and a smaller increase in area (38.4 ? 106 mV-s) for the material eluting after 30 min (Fig. 4d). The major changes in compositions were the loss of a and b-casein and the increase in soluble proteins or peptides with retention time between 10 and 30 min. The compounds eluting between 10 and 30 min appears to be native k-casein and a mixture of modi?ed and or hydrolyzed caseins. Based on the results from SDSPAGE, the large broad peak with retention time 41.7min should contain only trace amounts of b-casein (Fig. 4d). Since the separation was performed on a reversed phase column, the compounds eluting after 30 min would be more hydrophobic in nature. These compounds along with those eluting earlier may represent a

highly functional protein fraction formed by heat-treating casein molecules near their pI. 3.6. CD analysis To determine if a conformational change occurred in sodium caseinate following heat treatment at low pH, the far-UV CD spectra were obtained at pH 3.0, 3.75, 8.0 and 6.78 (control). The results at pH 3.75 revealed an increase in beta sheet content from 15.19% (control) to 22.85% (Table 4). It is interesting to note that the structural changes were similar at all three pH values but the increase in hydrophobicity occurred only in the low pH range (Fig. 2a and b). The conformational changes at pH 3.75 may have exposed a greater number of hydrophobic groups to the surface to produce the observed increase in hydrophobicity. Both the b-sheet and random structure changed at pH 3, 3.75 and 8.0, whereas the a-helix content remained constant (Table 4). Results from SDS-PAGE showed a decrease in both as1- and b-caseins following heat treatment at pH 3.75 and an increase in a band eluting with k-casein (Fig. 4). Studies on b-casein secondary structure revealed no evidence of a-helix structure (Chaplin, Clark & Smith, 1988) due to the high number of proline residues in the molecule. Studies on the threedimensional structure of k-casein (Kumosinski, Brown & Farrell, 1993), showed two sets of antiparallel b-sheet structures containing predominantly hydrophobic side chains. Because of its higher pI (5.3±5.8) values (Eigle et al., 1984), k-casein would be more soluble at pH 3.75 than a- or b-caseins (pI 4.2 and 4.7 respectively, Eigle et al.), but would not be present in large amounts in the soluble fraction due to its low concentration in the original sodium caseinate. It is not clear whether a- or b-casein or both hydrolyze as a function of pH and heat treatment. These conformational and compositional changes may be responsible for the observed increase in hydrophobicity and absorbency at 280 nm. Although the changes in b-sheet at pH 3 and 8 were similar to those at pH 3.75, the physicochemical and functional properties were di?erent. It is important to note that all the proteins at pH 3.0 and 8.0 were soluble giving those samples a much di?erent composition. 3.7. Protein functional properties 3.7.1. Emulsifying activity index The e?ectiveness of food proteins as emulsi?ers is commonly measured and expressed as EAI, which measures the turbidity of an emulsion at a single wavelength (Lee et al., 1992). The EAI of the soluble protein fractions from pH and heat-treated caseinate are shown in Fig. 5. EAI was temperature independent with a marginally signi?cant pH*pH interaction (P<0.077). The maximum EAI occurred around pH 3.5±4.0 (on a per

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Fig. 4. RP-HPLC elution pro?les of the soluble protein fractions; (a) pH 6.78; control (b) pH 3.75, no heat treatment; (c) pH 3.75, at 92.5 C and 10 min; (d) pH 3.75, at 92.5 C and 20 min. Table 4 Secondary structure of the soluble protein fractions after heat treatment at 100 C for 5 min Structure Helix b-Sheet Turn Random pH 6.78 (control) 21.70 15.19 23.52 39.56 pH 3.75 22.04 22.85 22.36 32.37 pH 3.00 23.19 24.31 20.94 31.14 pH 8.00 20.95 24.02 21.66 33.20

gram soluble protein basis) and the observed conformational changes and increases in hydrophobicity were also detected in this pH range suggesting a possible relationship. The properties of the soluble proteins near the pI appear to be very di?erent from those at other pHs. 3.7.2. Emulsifying capacity Emulsifying capacity is a measure of the maximum amount of oil that can be emulsi?ed under speci?ed

conditions by a unit weight of protein. The emulsifying capacity of the soluble protein fractions (Fig. 6) were signi?cantly related to pH (P<0.0005) and pH*pH (P<0.0001) interactions and independent of temperature. However, the maximum EC (on a per g soluble protein basis) was at pH 3.5±4 and the minimum was at pH 7±8. Again it appears that the functional properties of the soluble proteins are related to the observed conformational and hydrophobicity changes. 3.8. Relationships between physico-chemical and functional properties The physicochemical properties which directly a?ect the functional behavior of proteins are ultimately related to their structure (Damodaran, 1996). To better understand the structural±functional relationship of sodium caseinate, correlations between protein solubility

F. Jahaniaval et al. / Food Research International 33 (2000) 637±647

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studies would also show a positive correlation if the initial protein concentration (0.5%) was used in the EAI calculation (r=0.92; P<0.001) rather than soluble protein concentration. The calculated EAI would decrease near pH 3.5±4 due to the total protein concentration. When calculated on a per gram soluble protein basis, the EAI values maximize near pH 3.5±4, indicating that the soluble proteins near the pI have greater emulsi?cation properties than at other pHs. Sodium caseinate solubility and emulsifying capacity were also negatively correlated (r=?0.69; P<0.009). Again the soluble proteins near pH 3.5±4 have greater emulsifying capacity on a per gram soluble protein basis. 3.10. Hydrophobicity
Fig. 5. Emulsifying activity index of the soluble protein fractions as a function of pH and heat treatment.

Fig. 6. Emulsifying capacity of the soluble protein fractions as a function of pH and heat treatment.

and hydrophobicity with EAI and EC were measured using the SAS PROC CORR program. 3.9. Solubility and functional properties Protein solubility has been reported to be the most important factor in determining functionality (Kinsella, 1976). A signi?cant but negative correlation between solubility and EAI was observed (r=?0.905; P<0.001). As the solubility of the heat-treated caseinate reaches a minimal near pH 3.5±4, the EAI value approaches a maximum. In a similar study by Lieske and Konrad (1994), a positive correlation between protein solubility and EAI was shown at pH 2±8. EAI was minimal at pH 4±5 and increased above and below this pH range. Our

There was a strong correlation between ANS and CPA-hydrophobicity (r=0.98; P<0.001), indicating that more aliphatic and aromatic hydrophobic amino acid residues were exposed to the surface as a function of pH and temperature. Results from the hydrophobicity data (Fig. 2a and b) show that at pH 3±3.75 (where sodium caseinate has low solubility), both aromatic and aliphatic hydrophobicities are high. The EAI and EC of the soluble proteins increased at pH 3±3.75 (Figs. 5 and 6) as hydrophobicity increased. Many studies have reported a correlation between hydrophobicity and functionality of food proteins, such as emulsifying capacity (Kato & Nakai, 1980), interfacial tension and emulsifying activity index of k-casein (Nakai, 1983), foaming capacity (Townsend & Nakai, 1983) and emulsifying properties (Voutsinas, Nakai & Harwalker, 1983). Our study showed that protein functionality and hydrophobicity has a positive and signi?cant correlation. ANS hydrophobicity showed signi?cant correlation with EAI (r= 0.65; P<0.0146) and marginally with EC (r=0.53; P<0.057). CPA hydrophobicity showed marginally signi?cant correlation with EAI (r =0.55; P<0.053). 4. Conclusion The physicochemical properties of sodium caseinate were a?ected by pH and in some case the interaction between pH and temperature. Sodium caseinate has poor solubility near its pIs but the physicochemical properties of the soluble fraction increased dramatically. This property was not observed at pH (6±8) and pH 3, where the protein has higher solubility. The absorbency at 280 nm, hydrophobicity and CD analysis indicated structural changes at pH 3.5±4.0. The OPA and RP-HPLC results suggested that mild proteolysis was proceeding and may have initiated physicochemical

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