J. Agric. Food Chem. 1996, 44, 3437−3443
3437
Improved Emulsifying Properties of β-Barrel Domain Peptides Obtained by Membrane-Fractionation of a Limited Tryptic Hydrolysate of β-Lactoglobulin Xiaolin L. Huang, George L. Catignani, and Harold E. Swaisgood* Southeast Dairy Foods Research Center, Department of Food Science, North Carolina State University, Raleigh, North Carolina 27695-7624
Fragments of the β-barrel domain of β-lactoglobulin were obtained by membrane fractionation of a limited proteolysate prepared with an immobilized trypsin bioreactor. Analysis of this fraction by size-exclusion chromatography under physiological conditions indicated that the fraction contained a predominant peptide (50%) with a size of 8400 Da and several other peptides with sizes ranging from 2000 to 30 700 Da. Analysis of reductively denatured peptides by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol indicated the presence of a major peptide with a size of 6400 Da, suggesting that a small peptide was linked to the 8400-Da peptide by a disulfide bond. Comparison of the surface and emulsifying properties of the peptide fraction with those of intact β-lactoglobulin indicated that the domain peptides have a lower surface hydrophobicity and a slightly higher surface and interfacial tension. Furthermore, the emulsifying activity index for the domain peptides was twofold larger than that of the intact protein. Examination of the emulsion by scanning electron microscopy revealed that the oil droplets formed with the domain peptides were smaller than those formed with intact protein. Keywords: β-Lactoglobulin; tryptic hydrolysate; limited proteolysis; immobilized trypsin; emulsifying properties; β-barrel domain INTRODUCTION
Because of their amphipathic nature, proteins adsorb at oil/water interfaces, thereby lowering the surface tension and retarding coalescence of the droplets of the dispersed phase. The emulsifying activity of a protein depends on molecular properties, such as size and amphiphilicity, solubility, structural flexibility, charge distribution, and surface hydrophobicity, and on environmental factors, such as ionic strength, pH, temperature, and the nature of the solvent. Kinetic studies indicate that formation and stabilization of an oil-inwater emulsion are governed by the overall ability of the protein to diffuse to the oil/water interface, adsorb, unfold, rearrange, and spread to form a continuous cohesive film at the interface (Phillips, 1981; Kinsella and Whitehead, 1989). In general, those proteins with a flexible structure display a better emulsifying activity than those with a rigid structure (Phillips, 1981; Shimizu et al., 1981; Kato et al., 1985). Because limited proteloysis modifies the emulsifying properties of proteins, the effect of such treatment of whey proteins, particularly β-lactoglobulin (β-Lg), has been examined as a potential means for improvement of this functional property. Jost and Monti (1982) found that an oligopeptide preparation obtained by ultrafiltration of a tryptic hydrolysate of whey had improved emulsifying properties, and the emulsions exhibited more creaming stability. Treatment of whey proteins with trypsin, pepsin, or chymotrypsin yielded hydrolysates with improved foaming and emulsifying properties and higher emulsion stabilities compared with untreated proteins (Lakkis and Villota, 1990). However, detrimental effects of proteolysis of whey proteins on their emulsifying properties have also been reported. * Author to whom correspondence should be addressed. S0021-8561(96)00038-6 CCC: $12.00
For example, Kuehler and Stine (1974) observed that limited proteolysis of whey with prolase, pepsin, or pronase resulted in improved foaming ability but caused a decrease in emulsifying activity and emulsion stability. Moreover, a recent study by Turgeon et al. (1991) revealed that treatment of whey protein concentrate with trypsin or chymotrypsin caused a decrease in emulsifying capacity when the total hydrolysate was used; however, after removal of free amino acids and small peptides by ultrafiltration, an oligopeptide fraction was obtained that exhibited improved emulsifying capacity. In a subsequent study (Turgeon et al., 1992), a similar result was obtained with a β-Lg hydrolysate; however, fractionation of the hydrolysate yielded an oligopeptide, β-Lg(41-60), with a size of 2300 Da that possessed improved surface activity properties and emulsifying capacity. The reasons for these contradictory results are not clear; however, the characteristics of the substrates and enzymes used and, especially, the extent of hydrolysis may play an important role. As the major protein component of whey, β-Lg is responsible for much of the functional behavior (Mulvihill and Kinsella, 1987). The three-dimensional structure of the crystal form of β-Lg reveals that the core of the molecule consists of eight strands of anti-parallel β-structure that form a calyx with a short length of R-helix on the surface (Monaco et al., 1987). It has been suggested that the core β-barrel domain is the hydrophobic region that binds small hydrophobic molecules, such as fatty acids and retinol (Wishnia and Pinder, 1966; Monaco et al., 1987; Chen et al., 1993; Cho et al., 1994). In previous studies, we have shown that major fragments of the β-barrel domain can be produced by limited tryptic hydrolysis (Chen et al., 1993; Huang, et al., 1994). Characterization of the 7.9 and 8.4 kDa oligopeptides revealed that much of the original β-barrel structure was retained, although the structural stability © 1996 American Chemical Society
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was lower than that of the intact protein. Thus, these domain fragments may have unique structures with increased flexibility. In this study, β-Lg was subjected to limited proteolysis with an immobilized trypsin bioreactor under conditions that optimize production of these domain fragments, and these fragments were subsequently isolated by membrane-fractionation technology. The emulsifying properties of these oligopeptides were compared with those of intact β-Lg and egg white protein. MATERIALS AND METHODS Chemicals. All chemicals were of reagent grade, and distilled-deionized water was used throughout. Preparation of Celite Derivatives. Celite beads (R648, 30/50 mesh, Celite Corp., Lompoc, CA) were silanized by the method of Janolino and Swaisgood (1982). Surface amino groups were assayed quantitatively by the o-phthalaldehyde (OPA) method of Janolino and Swaisgood (1992). Aminopropyl Celite beads were subsequently succinylated by the nonaqueous method of DuVal et al. (1984). The completion of the reaction was determined by quantifying the disappearance of amino groups by the OPA method with aminopropyl beads as a reference. Immobilization of Trypsin. Trypsin (type XIII, TPCK treated, from bovine pancrease, Sigma) was immobilized on succinamidopropyl Celite (SAPC) beads at 4 °C by the method of Taylor (1979). Deaerated SAPC beads (100 mL) were activated for 30 min with 500 mL of 0.02 mM 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide (EDC) at pH 7.0. Then, 500 mL of trypsin solution (4 mg/mL in distilled-deionized water containing 0.04 M CaCl2, pH 7.0) was added to the mixture and allowed to react for 24 h. After reaction, the immobilized enzyme was washed with 2 M urea in phosphate buffer, pH 7.5, containing 0.02% NaN3 to remove noncovalently bound enzyme. Finally, the immobilized enzyme was washed with the phosphate buffer and stored at 4 °C. The activity of the immobilized trypsin was determined according to Taylor and Swaisgood (1980) with p-tosyl-Larginine methyl ester (TAME) as the substrate. The estimated activity was 24.6 µmol min-1 mL-1 of beads. Preparation of β-Lactoglobulin. Bovine β-Lg was purified from milk according to the method of Fox et al. (1967). Fifty-seven liters of skim milk, obtained from the North Carolina State University Dairy Plant, was adjusted to pH 4.6 with 4 M HCl, and 115 g of CaCl2 were added. After allowing the milk to stand for 6 h at 15 °C, the casein precipitate was removed by filtration through four layers of cheesecloth, and protein in the resulting whey was concentrated fivefold with a Millipore 10-kDa retention membrane. Trichloroacetic acid (TCA; 3%, w/v) was added to the concentrated whey solution to precipitate all proteins except β-Lg. The resulting precipitate was removed by centrifugation at 10000g for 20 min at 4 °C. The supernatant solution was adjusted to pH 2-3 with 1 M NaOH, concentrated, and dialyzed against distilled-deionized water, with the Millipore Concentrator/Dialyzer system, until the pH reached 6-7. The concentrated solution was lyophilized, and the protein powder was stored at -22 °C. The purity of the preparation was estimated to be >96% as determined by size-exclusion chromatography. Preparation of the Oligopeptide Fraction. The bioprocess combining the immobilized trypsin bioreactor with membrane fractionation of the products is illustrated in Figure 1. Four hundred milliliters of β-Lg solution (5 mg/mL in 10 mM ammonium acetate buffer, pH 7.6) were recirculated through a fluidized-bed bioreactor containing 100 mL of immobilized trypsin for 60 min at 24 °C. The resulting limited hydrolysate was first fractionated with a 30-kDa retention membrane (YM30, Amicon, Inc., Beverly, MA), and the resulting permeate was further fractionated with a 3-kDa retention membrane (YM3, Amicon); both processes were at 4 °C. The retentate from the YM3 membrane, representing the domain fragment oligopeptides, was lyophilized, and the powder was stored at -22 °C.
Figure 1. Schematic illustration of an immobilized enzymemembrane fractionation bioprocess for preparation of the domain fraction of β-Lg. Determination of the Degree of Hydrolysis. The degree of hydrolysis was determined by measuring the increase in concentration of amino groups resulting from proteolysis. Lyophilized powder from 50 mL of the hydrolysate and lyophilized native β-Lg as a reference were dissolved in 40 mM imidazole buffer, pH 7.5, and the fluorescence intensities were measured after the OPA reaction. The fluorescence of a 0.1µg/mL solution of quinine sulfate was used as a reference to calculate the relative fluorescence intensity (RFI). The calculated RFI was converted to amino group concentration with a specific RFI value of 2.71 × 108/mol of lysyl residues in β-Lg (Goodno et al., 1981). The degree of hydrolysis was calculated from the increase in amino group concentration by the relationship
DH(%) ) Nt/N0 × 100
(1)
where Nt is the increase in concentration of amino groups due to proteolysis at time t and N0 is the total amino group concentration from all residues in the protein if complete hydrolysis occurred (both values are expressed as mol NH2/g of protein). Determination of Protein Concentration. The concentration of soluble protein was assayed by the dye-binding method of Bradford (1976) with bovine serum albumin (BSA, Sigma) as a standard. Fast-Protein Liquid Chromatography (FPLC) SizeExclusion Chromatography. Molecular weights of the oligopeptides in the hydrolysate and in the isolated fractions were estimated by size-exclusion chromatography with a Superose TM HR 10/30 column. The FPLC system used consisted of a model 510 pump, a model UK6 injector, and a model 990 photodiode array detector (Waters, Milford, MA). The detector was equipped with an APC IV series computer (NEC Information System, Inc., Foxborough, MA) for data acquisition and spectral analysis. The elution buffer was 20 mM Tris-HCl, pH 7.0, containing 150 mM NaCl and 0.02% NaN3. BSA (66 000 Da), β-Lg (dimer, 30 000 Da), R-lactalbumin (14 200 Da), and vitamin B12 (1355 Da) were used as the molecular weight standards. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The domain oligopeptide fraction was analyzed by SDS-PAGE using an LKB 2050 Midget Electrophoresis unit (LKB Produkter AB, Bromma, Sweden) with a gradient gel of 16-27% acrylamide (Huang et al., 1994). The protein or peptide bands were visualized by staining with Coomassie brilliant blue R250. Measurement of Surface Hydrophobicity. Surface hydrophobicity was determined according to the method of Kato and Nakai (1980) with 1-anilino-8-naphthalenesulfonate
J. Agric. Food Chem., Vol. 44, No. 11, 1996 3439
Emulsifying Properties of β-Barrel Domain Peptides from β-Lactoglobulin
Figure 2. Typical elution profiles of limited tryptic hydrolysates of (A) β-Lg and (B) 3-kDa membrane retentate (domain fraction) obtained from size-exclusion chromatography (Superose TM HR 10/30). The elution buffer was 20 mM Tris-HCl, pH 7.0, containing 15 mM NaCl and 0.02% NaN3. Molecular weight markers were (1) bovine serum albumin (MW ) 66 000), (2) β-Lg (dimer, MW ) 36 700), (3) R-lactalbumin (MW ) 14 200), and (4) vitamin B12 (MW ) 1355). The markers were chromatographed individually. (ANS, Sigma). Each protein sample (2 mL) was serially diluted with 10 mM phosphate buffer, pH 7.0, to give protein concentrations ranging from 0.001 to 0.03% (w/v). Ten microliters of ANS solution (8.0 mM in phosphate buffer) were added to 2 mL of the diluted protein solutions, and the fluorescence intensities (FI) were measured with a spectrofluorometer (Optical Technology Devices, Inc., Elmford, NY) at excitation and emission wavelengths of 390 and 470 nm, respectively. The 30% FI value of ANS in methanol was used as a reference value for calculating the RFI of the proteinANS solutions. The RFI for the protein-ANS interaction was obtained by subtraction of the RFI of a control protein solution without ANS. Slopes of plots of RFI versus protein concentration, designated S0, were calculated by linear regression. Measurement of Surface and Interfacial Tension. Surface tension of 0.5% (w/v) protein solutions in 10 mM phosphate buffer, pH 7.0, was measured at 24 °C with an interfacial tensiometer (CSC NOS70545, CSC Scientific Company, Inc., Fairfax, VA) equipped with a 55 × 28-mm glass plate. The interfacial tension between the protein solution and peanut oil was also measured under similar conditions. The tensiometer was calibrated, and the plate was cleaned according to instructions provided by the manufacturer. Determination of the Emulsifying Activity Index. The emulsifying activity index (EAI) was determined according to the turbidimetric method of Pearce and Kinsella (1978). Pure peanut oil (0.5 mL, Sigma) was dispersed in 1.5 mL of protein solution (0.5% w/v in 10 mM phosphate buffer ranging in pH from 3.0 to 9.0) in a 13 × 100-mm culture tube with a Tissumizer (Tekmar Company, Cincinnati, OH) operated at 12 000 rpm for 1 min. Aliquots (5 mL) of the emulsions were immediately diluted 1000-fold with 0.1% SDS solution in 10 mM phosphate buffer, pH 7.5, and the turbidity was measured at 500 nm. The EAI was calculated with the relationship
EAI ) 2τ/φC
(2)
where turbidity τ ) 2.303 A500/l, l is the cuvette pathlength, φ is the volume fraction of the oil phase, and C is the protein concentration.
Determination of Emulsion Stability. Stability of the protein emulsions was evaluated according to the method of Britten and Giroux (1991) with some modifications. Emulsions prepared as just described were incubated and agitated in a temperature-controlled shaker (37 °C, 150 rpm) to facilitate and increase collisions between emulsion droplets, thus accelerating coalescence. Aliquotes (5 µL) of the emulsion were withdrawn from the bottom layer at various times and diluted 500 to 1000-fold with 0.1% SDS solution, and the turbidity was determined at 500 nm as described for EAI measurements. Because coalescence results in a decrease in turbidity with time, τ0/τt was plotted as a function of time, where τ0 is initial the turbidity and τt is the turbidity at time t. The slope of this linear plot obtained by least squares linear regression was used as an index of the stability. Scanning Electron Microscopy (SEM). Samples for SEM were prepared as described by Liboff et al. (1988). Freshly prepared emulsions made with β-Lg or the domain oligopeptide fraction (0.5% w/v) were gently mixed (1:1) with a warm 1% agarose solution. After gentle stirring with a wooden stick, the dispersion was allowed to gel at 24 °C. The resulting gels were cut into cubes (=1 mm3) and fixed with 4% glutaraldehyde in 0.1 M phosphate buffer, pH 7.0, overnight. The fixed gels were dehydrated with a graded series of methanol solutions, critical point dried, mounted on aluminum stubs, coated with gold/palladium, and photographed at various magnifications in a ETEC autoscan microscope (SEM 505 T, Phillips, Holland). Duplicate samples were analyzed with the selection of at least five different fields for photography. RESULTS
Preparation of β-Barrel Domain Fragments. β-Lg was subjected to limited proteolysis with the immobilized trypsin bioreactor at 24 °C for 60 min. A typical elution profile from FPLC size-exclusion chromatography of the limited hydrolysate is shown in Figure 2A. Analyses of the data from chromatography
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Table 1. Molecular Size Distribution of the 3-kDa Membrane-Fractionated Hydrolysate of β-Lactoglobulin As Determined by Size-Exclusion HPLCa peak area (%) molecular weight (Da)
retentate
permeate
30 700 16 400 8 400 4 700 3000) 84
a Mean of duplicate measurements; the elution profile was monitored at 214 nm, and the percentage of each peak represents the ratio based on the total area of all peaks.
Figure 3. SDS-PAGE of β-Lg and the domain fraction in the presence of 2-mercaptoethanol. Lane 1, protein markers; lane 2, native β-Lg (5 µg); and lanes 3 and 4, the domain fraction (4 and 12 µg, respectively).
and OPA assays indicated that ∼55% of the original β-Lg was hydrolyzed with a degree of hydrolysis (DH) of 2.4%. The remaining 45% of intact protein was retained by the 30-kDa membrane and could be recycled through the bioreactor. Membrane fractionation of the hydrolysate, as outlined in Figure 1, yielded the domain fragments in the 3-kDa membrane retentate. Estimation of the molecular size by size-exclusion chromatography under physiological conditions indicated peptides ranging from
