Article pubs.acs.org/JAFC
Emulsifying and Emulsion-Stabilizing Properties of Gluten Hydrolysates Iris J. Joye*,†,‡ and David J. McClements† †
Biopolymers and Colloids Research Laboratory, Department of Food Science, University of Massachusetts, Amherst, Massachusetts 01003, United States ‡ Laboratory of Food Chemistry and Biochemistry, KU Leuven, Leuven, Belgium ABSTRACT: Gluten is produced as a coproduct of the wheat starch isolation process. In this study, gluten was hydrolyzed to degrees of hydrolysis (DH) of 3−6−10 and 1−2−3 with alcalase and trypsin, respectively. These peptidases have a clearly distinct substrate specificity. Corn oil-in-water emulsions (10 wt % oil) were prepared by high-pressure homogenization at pH 7.5. Gluten peptides with DH 3 proved to be the most effective in producing peptides displaying emulsifying properties. Higher levels of alcalase hydrolysates (2.0 wt %) than of trypsin hydrolysates (1.0 wt %) were required to produce stable emulsions with small droplet sizes, which is attributed to differences in the nature of the peptides formed. The emulsions had small mean droplet diameters (d32 < 1000 nm). Emulsions produced with trypsin hydrolysates (stable after 9 days at 55 °C) displayed better thermal stability compared to those produced with alcalase hydrolysates (destabilized after 2 days at 37 °C). The hydrolysate-containing emulsions, however, were quickly destabilized by salt addition (≤100 mM NaCl) and when the pH approached the isoelectric point of the coated droplets (pH ∼5.5). Microscopic analysis revealed the formation of air-in-oil-in-water emulsions at lower hydrolysate concentrations, whereas at higher concentrations (≥3.0 wt %) extensive flocculation occurred. Both phenomena contributed to creaming of the emulsions. These results may be useful for the utilization of gluten hydrolysates in food and beverage products. KEYWORDS: gluten, hydrolysates, emulsifier, emulsion stabilization
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INTRODUCTION Gluten protein makes up 80% of wheat flour protein. It consists of monomeric gliadin and polymeric glutenin.1 Wheat gluten is a coproduct of the wheat starch industry. Nowadays, the main application of isolated gluten is as a dough and bread improver in the breadmaking industry. It contains high levels of glutamine and nonpolar amino acids (proline and glycine), which render the protein insoluble in aqueous media.2 The low water solubility of gluten at neutral pH limits its application as a functional ingredient in the food industry, even though its price is low compared to that of other protein isolates, such as those from pea, soy, or animal sources.3 Several chemical and enzymatic modification strategies for improving the water solubility of gluten have been explored. Enzymatic hydrolysis alters the molecular weight and the exposure of hydrophobic regions to the surrounding aqueous phase.4 The resulting peptides can, hence, display functional and bioactive features that are very different from those of the original proteins.5−9 Traditionally, egg yolk has been used as an emulsifier in food emulsions such as mayonnaise and salad dressings. The emulsifying capacity of plant proteins is promising for replacing egg yolk in these products and the production of lowcholesterol and Salmonella-free food emulsions.10 The increasing interest in the utilization of plant proteins as functional ingredients is also driven by the search to valorize these underutilized and often cheap proteins. Indeed, gluten proteins can be purchased at prices that are 2−7-fold lower than the price normally paid for other protein sources widely used in the food industry, such as gelatin, whey protein isolate, and casein. To date, several studies3,6,10−15 have explored the emulsifying © 2014 American Chemical Society
properties of gluten protein hydrolysates. Most of these studies used relatively simple homogenization methods, such as highspeed blending, with various levels of success. In the current work, two peptidases [alcalase (Alc) and trypsin (Try)] with distinct hydrolysis behaviors have been studied to increase the solubility and emulsification properties of gluten. Whereas Try cleaves at a few specific sites in the amino acid chain,16 Alc has a much broader substrate specificity.17 These two peptidases, hence, offer the opportunity to produce gluten hydrolysates with diverse molecular properties. The objective of the present study was to analyze the potential of gluten hydrolysates produced using these two peptidases for forming and stabilizing emulsions prepared using a high-pressure homogenization procedure (microfluidization). The information obtained should provide insights into the optimum molecular properties required for gluten hydrolysates, thereby facilitating the rational design of peptidase choice and hydrolysis conditions.
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MATERIALS AND METHODS
Chemicals. All chemicals, reagents, and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were of analytical grade unless stated otherwise. Proximate Analysis. The moisture content of gluten and freezedried gluten hydrolysates was measured as described in AACC Received: Revised: Accepted: Published: 2623
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Approved Method 44-15.02.18 The protein (N × 5.7) content was measured using a Dumas combustion method, adapted from the AOAC Official Method19 to an automated Dumas protein analysis system (EAS, VarioMax N/CN, Elt, Gouda, The Netherlands). Gluten Hydrolysis. Gluten was hydrolyzed with porcine pancreatic Try (EC no. 3.4.21.4, Sigma-Aldrich) or Alc 2.4 L FG (EC no. 3.4.21.62, Novozymes, Bagsvaerd, Denmark). An aqueous protein dispersion was prepared by dispersing 6.0 wt % commercial gluten (protein content on dry basis of 83.3%, Syral Belgium, Aalst, Belgium) in water. The dispersion was then heated to 50 °C (Try) or 60 °C (Alc) in a water bath under continuous stirring prior to manual adjustment of the pH by addition of NaOH (1.0 M) to pH 8.0 (Try) or 10.0 (Alc). After the desired temperature and pH conditions were obtained, the peptidase was added and gluten hydrolysis was monitored using the pH-stat method.20 The degree of hydrolysis [DH, defined as the number of peptide bonds hydrolyzed (h) over the total number of peptide bonds per unit weight present in gluten (htot)] was controlled by varying the hydrolysis time. It was calculated from the amount of base needed during hydrolysis to maintain a constant pH in the hydrolysis reaction medium:
measured by static light scattering (Mastersizer 2000, Malvern Instruments, Malvern, UK). The droplet charge (ζ-potential) was determined by particle electrophoresis (Zetasizer Nano ZS series, Malvern Instruments, Worcestershire, UK). Prior to droplet charge analysis, samples were diluted 100-fold with 10 mM Tris-HCl buffer to avoid multiple scattering effects. Emulsion Microstructure. The microstructure of the emulsions was visualized using optical and/or confocal scanning laser microscopy with a 60× objective and a 10× eyepiece (Nikon Eclipse 80i, Nikon Instruments Inc., Melville, NY, USA). A small aliquot (about 8 μL) was placed on a microscope slide and covered with a coverslip prior to analysis. Prior to emulsification, the oil and the aqueous phase of the emulsion samples used for confocal microscopy were stained with Nile Red [400 μL of Nile red (0.1% (w/v) solution in ethanol) on 7.5 g of oil] and fluorescein isothiocyanate [FITC, 800 μL of FITC (0.1% (w/ v) solution in dimethyl sulfoxide) on 67.5 g of aqueous phase] to visualize lipids and proteins, respectively. The emissions of Nile Red and FITC were detected in the 605 and 515 nm channels, respectively, equipped with long pass filters (HQ 605LP/75 m and HQ 515/30). The microstructure images were analyzed using image analysis software (Nikon). Emulsion Stability. Stability to Thermal Processing. Freshly prepared emulsions were distributed over different glass test tubes and incubated for 30 min in water baths set at different temperatures (30− 90 °C). The samples were subsequently allowed to cool to 20 °C, and the particle size distribution was analyzed as described above 24 h and 9 days after the heat treatment. Stability to Isothermal Storage. Freshly prepared emulsions were distributed over different glass test tubes and immediately incubated for 9 days in temperature-controlled chambers set at different temperatures (4, 20, 37, and 55 °C). Simultaneously, parallel samples containing 150 mM NaCl were stored under the same conditions. The ionic strength was adjusted by the addition of highly concentrated NaCl solution (6.0 M). The final concentration of oil in all of the emulsions was kept constant by adjusting the total volume of the emulsions using water. The particle size distribution of the samples was analyzed as described above 1, 2, 3, 6, and 9 days after preparation. Stability to Different pH Conditions. Freshly prepared emulsions were distributed over different glass test tubes, and their pH values were immediately adjusted by adding NaOH (1.0 M) or HCl (1.0 M) to obtain values ranging from pH 3.0 to 9.0. The particle size distribution of these samples was determined 24 h and 9 days after adjustment of the pH. Stability to Different Ionic Strength Conditions. The ionic strength of freshly prepared emulsions was altered by adding a highly concentrated NaCl solution (6.0 M) to produce samples containing 0.0−0.5 M NaCl. The final concentration of oil in all of the emulsions was kept constant by adding water to adjust their total volume. The particle size distribution of these samples was determined 24 h and 9 days after adjustment of the ionic strength. Statistical Analysis. All measurements were performed on at least three freshly prepared samples (except for the preliminary experiments in which the best-performing hydrolysates were determined) and were reported as means and standard deviations, analyzed using a spreadsheet statistical package (Microsoft Excel 2011). The data were analyzed with Tukey’s tests (P value 3.0 wt % for Alc3 and >1.0 wt % for Try3). In most cases, the droplet size decreased with increasing hydrolysate concentration until a minimum value was reached (Figure 2a,b), which is in agreement with the behavior expected for emulsifiers during high-pressure homogenization.22 However, anomalous behavior was observed for the emulsions prepared with high levels of Try3 hydrolysates,
Figure 1. Influence of degree of hydrolysis (DH) on mean particle diameter of emulsions (d32) made with 1.0 wt % (a) and 2.5 wt % (b) of gluten hydrolysates produced with alcalase (Alc) and trypsin (Try). The degrees of hydrolysis of the gluten hydrolysates produced with Alc were 3 (Alc3), 6 (Alc6), and 10 (Alc10), whereas those of the hydrolysates produced with Try were 1 (Try1), 2 (Try2), and 3 (Try3). All emulsions contained 10 wt % corn oil. The reported values were recorded 3 h and 7 days after emulsion production. a−d and A−F represent Tukey groups with p < 0.05 for comparison of the particle diameter as a function of peptidase/DH of 3-h- and 7-day-old emulsions, respectively. ∗ Upon 7 days of storage, the emulsion produced with 1.0% Alc3 hydrolysate displayed extensive creaming, which hindered droplet diameter measurements.
where a steep increase in particle size was observed at hydrolysate concentrations >2.0 wt %. On the basis of particle size distribution measurements of the fresh and 7-day-old emulsions (Figure 2a,b), 1.0% Try3 and 2.0% Alc3 were selected as the best-performing hydrolysate concentrations in terms of both emulsion formation and stabilization. Microscopic analyses confirmed the bimodal droplet size distribution, as well as flocculation of emulsion droplets produced with higher hydrolysate concentrations (Figure 2c). To obtain an estimation of the saturation concentration of the hydrolysates at the droplet interface, the protein content in the aqueous phase of the emulsions was determined and compared to the protein content in the aqueous phase prior to emulsification. This ratio was plotted as a function of increasing hydrolysate concentration (Figure 3). It was expected that for the lowest hydrolysate concentrations, no residual protein or very low protein concentrations would be found in the aqueous 2625
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Figure 3. Influence of gluten hydrolysate concentration on the percentage of nonadsorbed proteins found in the aqueous continuous phase of 10 wt % corn oil emulsions. Emulsions were produced using gluten hydrolysates with a degree of hydrolysis of 3 made with alcalase (Alc3) and trypsin (Try3).
hydrolysate addition level.22 However, no such result was obtained (Figure 3). When the protein contents of the continuous aqueous phase of the emulsions were compared to those of the original aqueous phase prior to emulsification, it was clear that about 50 or 60% of the hydrolysates produced with Try or Alc, respectively, were not adsorbed at the droplet interface. This was largely independent of the initial protein concentration (Figure 3). The heterogeneity of the hydrolysate samples in terms of surface activity certainly contributes to this behavior. It is possible that a fraction of the hydrolysates was hydrophobic or surface-active (and therefore became associated with the oil droplets or were even solubilized in the oil phase), whereas another fraction was hydrophilic (and therefore remained in the aqueous phase). Nevertheless, further research using isolated fractions of the hydrolysates would be needed to establish this. The surface loads of the hydrolysates (i.e., the amount adsorbed per unit surface area) at saturation were estimated to be about 9 and 13 mg/m2 for Try3 and Alc3 hydrolystates, respectively. These values are on the high end of values typically reported for food proteins (around 1−10 mg/m2). However, these values should be interpreted carefully for a number of reasons. First, the hydrolysates were a mixture of different types of molecules, rather than a single type, which biases the determination of the exact protein concentration Furthermore, the transfer of part of the hydrolysates to the oil phase cannot be excluded. Second, the mean droplet diameter (d32) used in the calculations assumes that the droplets are not flocculated, although in reality some droplet flocculation may have occurred. For this reason, the surface load for the Try3 hydrolysate was calculated on the basis of the droplet size data at 2.0% of hydrolysate, before extensive flocculation occurred (Figure 2c). Third, there may have been some problems in making reliable protein concentration measurements in the samples due to light scattering by small emulsion droplets or protein aggregates. Emulsion Stability. Upon 24 h of storage of the original Alc3 and Try3 emulsions, some tendency toward creaming could already be observed. Emulsions containing oil droplets coated by either Try3 or Alc3 hydrolysates were highly sensitive to changes in ionic strength and pH (Figure 4). The electrical
Figure 2. (a, b) Influence of gluten hydrolysate concentration on the mean particle diameter (d32) of 10 wt % corn oil emulsions, freshly prepared and 7 days old. Emulsions were made using gluten hydrolysates with a degree of hydrolysis of 3 produced with alcalase [Alc3 (a)] and trypsin [Try3 (b)]. All emulsions contained 10 wt % corn oil. a−d and A−E represent Tukey groups with p < 0.05 for comparison of the particle diameter as a function of hydrolysate concentration of freshly made and 7-day-old emulsions, respectively. (c) Optical microscopic images of emulsions produced with increasing concentrations [i.e., 0.1 (i), 0.5 (ii), 1.0 (iii), 1.5 (iv), 2.0 (v), and 3.0 (vi) wt %] of gluten hydrolysates. The hydrolysates had a degree of hydrolysis of 3 and were produced with Try. All emulsions contained 10 wt % corn oil. The scale bars represent 20 μm.
phase, and from a critical hydrolysate concentration on the free protein concentration would increase as a function of increasing 2626
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However, extensive flocculation was observed in both types of emulsions using optical microscopy analysis. Thus, it is possible that the flocs in the Alc3 emulsions were disrupted during the dilution and mixing stages of the particle size analysis, which highlights the importance of using more than one method for analyzing aggregated systems. The Alc3 emulsions were relatively stable to droplet aggregation when stored at low temperatures (4 and 20 °C), but they exhibited considerable droplet aggregation behavior when stored at more elevated temperatures (i.e., 37 and 55 °C) (Figure 5a). Conversely, the Try3 emulsions appeared to be stable during isothermal long-term storage at all storage temperatures as no significant changes in particle diameter were observed (Figure 5b). Furthermore, in contrast to Alc3 hydrolysates, Try3 hydrolysates proved to be effective at stabilizing emulsions against droplet aggregation during shorttime thermal processing (30 min) (Figure 5c). Microstructure Analysis. Microscopic analysis of the emulsions not only confirmed the bimodal size distribution of the oil droplets but also showed the presence of intriguing structures within the larger emulsion droplets. We observed smaller droplets enclosed within the larger oil droplets (Figures 2b and 6a). To explore the nature of these smaller subdroplets, the oil and water phases were stained prior to emulsification. The fact that the internal droplets were not stained by either dye suggests that they did not contain lipid or protein and may therefore have been trapped air bubbles (Figure 6b). These airin-oil-in-water systems could be detected in all emulsions made with hydrolysate concentrations below 3.0 wt %. Above this concentration, no separate oil droplets could be observed. The strong creaming tendency of the emulsions after production is probably partially caused by the more pronounced density difference between the continuous and disperged phase due to the presence of air.
Figure 4. Emulsion characteristics in different pH and ionic strength conditions: (a) influence of pH on the ζ-potential of droplets of emulsions produced with gluten hydrolysates with a degree of hydrolysis of 3 made with alcalase (Alc3) and trypsin (Try3) (reported values were recorded 24 h after pH adjustment); (b) influence of ionic strength on mean particle diameter (d32) (reported values were recorded 24 h after ionic strength adjustment). The shaded bars represent the particle diameter of the freshly made emulsions prior to ionic strength adjustment. The influence of ionic strength on the appearance of the emulsions is shown in the pictures. Extensive creaming occurred at concentrations ≥0.1 M NaCl for the Alc3 and Try3 emulsions. a and b and A and B represent Tukey groups with p < 0.05 for comparison of the evolution of the particle diameter in function of ionic strength of Alc3 and Try3 emulsions, respectively.
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DISCUSSION Gluten can be divided into the two main storage protein types found in wheat kernels, that is, gliadin and glutenin. In an earlier study, Takeda and co-workers23 found that unhydrolyzed gliadin and glutenin have different emulsifying behaviors at low pH. Walstra22 found that peptides formed by hydrolyzing native proteins are more effective emulsifiers than the native proteins themselves, provided that they are not too small, they are water-soluble, and they are amphiphilic. Previous studies have shown the emulsifying capacity of gluten protein hydrolysates using relatively simple homogenization techniques.3,4,12,14 In most of these studies, however, no information on the droplet size distribution was reported. Linares and coworkers13 reported oil droplet sizes of about 20 μm for emulsions produced using a high-speed blender, which could be decreased about 10-fold when they were passed through a highpressure valve homogenizer.15 In the current study, the average droplet diameter could be decreased into the submicrometer range using a high-pressure homogenizer (microfluidizer) provided the emulsions were prepared at pH conditions sufficiently distinct from the point of zero charge. This is in accordance with what is observed for other proteins.24−26 The obtained particle diameters are comparable to or larger than those of emulsions made with whey protein isolate (0.27%), βlactoglobulin (0.05%), or sodium caseinate (0.27%) using similar production parameters.25−27 The gluten peptides formed by hydrolysis produced electrically charged droplets. The most likely stabilization mechanism preventing droplet
charge on the hydrolysate-coated oil droplets went from highly positive at low pH to highly negative at high pH, with a point of zero charge just above pH 5 (Figure 4a). At pH values around the isoelectric point of the droplets (pH ≈5), there was a large increase in mean particle size and extensive creaming was observed. At relatively high pH values (pH 8.0 and 9.0), the emulsions were relatively stable to droplet aggregation, maintaining relatively small droplet sizes (d32 < 1000 nm) after 9 days of storage (results not shown). At pH 3.0, the particle diameter of the Try3 emulsions remained relatively small, whereas Alc3 emulsions were much less stable and were prone to creaming. All emulsions produced were destabilized by salt addition and displayed extensive creaming behavior. The mean particle diameter of the emulsions produced with Try3 determined by light scattering significantly increased for NaCl ≥ 100 mM, indicating that salt addition promoted droplet aggregation. On the other hand, the particle size measured by light scattering appeared to be relatively small (d32 < 1000 nm) for the Alc3 emulsions at all salt concentrations (Figure 4c). 2627
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Figure 5. continued (Try3). Emulsions were heated for 30 min at the indicated temperatures before they were cooled and stored at 20 °C. Droplet sizes shown were recorded 24 h and 9 days after heat processing. a, b and A−C represent Tukey groups with p < 0.05 for comparison of the evolution of the particle diameter as a function of treatment temperature of freshly produced and 9-day-old Alc3 emulsions, respectively. a, b and A, B represent Tukey groups with p < 0.05 for comparison of the evolution of the particle diameter as a function of treatment temperature of freshly produced and 9-day-old Try3 emulsions, respectively.
aggregation is, hence, electrostatic repulsion.28−30 For example, at pH 8 and 9, the oil droplets had a strong negative charge (Figure 4a), which would generate a strong electrostatic repulsion that prevents droplet flocculation (Figure 4b). By increasing the ionic strength or decreasing the pH, the net negative charge on the droplets is shielded or decreased and the emulsion is destabilized by flocculation. This is a common phenomenon observed when using proteins as emulsifiers.31,32 The increase in net particle size due to droplet flocculation then leads to accelerated creaming. At present, a number of nutritional beverages or shakes exist that have a pH value near neutral in which the here-produced emulsion droplets coated by the hydrolysates could be used. Upon hydrolysis of gluten proteins, a whole range of peptides with largely varying molecular weight, hydrophobicity, and surface activity are created. Previous studies12,14 indicate that too extensive hydrolysis has an adverse effect on the hydrolysates’ emulsifying capacity. Conversely, Linares and colleagues13 did not find a correlation between degree of hydrolysis and emulsifying properties of solubilized gluten peptides. Previous research on the emulsifying properties of peptides produced from other protein sources, however, showed that there is indeed a minimum size, that is, about 2000 Da (∼20 amino acid residues), required for peptides to display emulsifying potential.33 However, the molecular weight is not the only factor determining the surface-active properties of peptides. Turgeon and co-workers33 described that the amphiphilicity of peptides also plays a key role and suggested that the hydrophobic residues should be grouped in discrete patches of at least three to five residues, which are separated by regions containing at least two to three polar residues. When evaluating the peptides that could possibly be formed by Try and Alc starting from randomly selected gluten protein sequences [i.e., a HMW glutenin subunit (GenBank accession no. ABQ14770.1), a LMW glutenin subunit (GenBank accession no. AAS10192.1), a ω-gliadin (GenBank accession no. AAT01617.1), and a γ-gliadin (GenBank accession no. AAQ63860)] and assuming that Try exclusively hydrolyzes the C-terminal peptide bonds of Arg and Lys16 and Alc displays a similar specificity on gluten proteins as has been described on casein,17 it was found that in all cases Try produces more peptides which meet the above listed requirements to be good emulsifiers. Indeed, Alc has a preference to hydrolyze peptide bonds that are N-terminal to hydrophobic residues and Cterminal to, among other residues, Leu and Tyr. As a result, only a small number of peptides containing the above-described hydrophobic patches will be formed from gluten proteins, as these patches, if present in the original protein, are largely cut into pieces during the hydrolysis process by Alc.
Figure 5. Emulsion stability to isothermal storage and heat processing. (a, b) Influence of isothermal storage on mean particle diameter (d32) of emulsions produced with gluten hydrolysates with a degree of hydrolysis of 3 made with alcalase (a) and trypsin (b). Emulsions were stored for up to 9 days. a, b; A, B; a−c; and A−D represent Tukey groups with p < 0.05 for comparison of the evolution of the particle diameter in function of storage time of emulsions stored at 4, 20, 37, and 55 °C, respectively. (c) Influence of heat processing on mean particle diameter (d32) of emulsions produced with gluten hydrolysates with a degree of hydrolysis of 3 made with alcalase (Alc3) and trypsin 2628
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Figure 6. Optical and confocal micrographs of an emulsion made with 0.1 wt % gluten hydrolysate with a degree of hydrolysis of 3 produced with trypsin. The oil phase (red) was stained with Nile Red, whereas the aqueous phase containing the gluten hydrolysates (green) was stained with fluorescein isothiocyanate. The black bubbles inside the oil droplets are assumed to be air. All emulsions contained 10 wt % corn oil.
surfaces and thus provided better protection against thermal destabilization and creaming of the emulsions upon heating. Furthermore, the heat treatment had probably already converted any free thiol groups on the gluten proteins to disulfide bridges, thereby limiting any additional protein crosslinking at high temperatures. This study has shown that gluten hydrolysates that display different emulsifying properties can be produced by careful selection of peptidase type and hydrolysis conditions. Of the two peptidases used, Try produced the most promising peptides with a DH of 3. The stability of the emulsions to environmental stresses (such as pH, ionic strength, and thermal processing) also depended on the peptidase type with which the hydrolysates were produced. Gluten hydrolysis may provide a valuable new source of emulsifiers for utilization in the food industry and could add value to byproducts created during starch isolation. The emulsifying properties of these hydrolysates may be further improved by fractionation of the complex peptide mixture. Furthermore, the capacity of the gluten peptides to produce air-filled emulsions holds promise for the production of reduced-fat products and food products with new functionalities. Further research is needed to optimize the conditions in which the latter can be produced.
When the protein contents of the continuous aqueous phase of the emulsions are compared to those of the original aqueous phase prior to emulsification, it is clear that about 50 or 60% of the hydrolysates produced with Try or Alc, respectively, are not adsorbed at the droplet interface. This seems to be largely independent of the initial protein concentration and indeed reflects the above-described difference between the two peptidases and confirms the production of peptides with largely varying surface-active properties. In addition, the higher nonadsorbed protein level for Alc3 emulsions partially explains the higher level of Alc3 hydrolysates relative to the level of Try3 hydrolysates needed to produce emulsions with similar droplet diameters. Furthermore, the high level of nonadsorbed peptides probably induces depletion flocculation at higher hydrolysate concentrations, that is, >1.0 and >2.0% for Try3 and Alc3, respectively. The steep increase in droplet size and creaming tendency as well as microscopic analysis of the emulsions produced with higher hydrolysate concentrations supports this hypothesis. Regardless of the hydrolysate concentration, all emulsions were relatively sensitive to creaming, which can be partially attributed to the presence of larger air-filled oil droplets (Figure 6). Previous studies already showed that gluten peptides are effective foaming agents.3,13,34 Furthermore, Takeda and coworkers23 showed that gluten, similar to one of its functions in dough, also stabilizes the air/water interface. We believe that during the production of the emulsions, air is incorporated into the liquid phase and that the gluten proteins stabilize these air bubbles in the aqueous phase. During homogenization in the microfluidizer, (part of) the air bubbles are forced into the oil droplets. As the interfacial corn oil/air tension (i.e., about 32 mN/m35) is lower than the interfacial hydrolysate solution/air tension (i.e., about 45 mN/m34), a system that is kinetically stable (but thermodynamically unstable) on the mesoscale (i.e., on the scale of emulsion droplets) is produced. Macroscale stability, however, is reasonably low as the increased density difference promotes creaming of the air-filled oil droplets. The Try hydrolysates displayed good heat stability, which is not commonly observed with many food proteins. Try and Alc hydrolysates underwent a heat treatment while inactivating the peptidase during their production. Consequently, they were already partially thermally denatured prior to homogenization. This first heat treatment is believed to minimize extensive denaturation of the peptides when adsorbed at the oil droplet
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AUTHOR INFORMATION
Corresponding Author
*(I.J.J.) Phone: +1 (413) 545-7157. Fax: +1 (413) 545-1262. Email:
[email protected]. Funding
We thank the U.S. Department of Agriculture, CREES, NRI, and AFRI Grants and the Massachusetts Department of Agricultural Resources for financial support of this work. I.J.J. gratefully acknowledges financial support from the Fonds voor Wetenschappelijk Onderzoek − Vlaanderen (FWO, Brussels, Belgium) and from the European Commission 7th Framework Program (FP7-People-2011-IOF-300408). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the Laboratory of Food Chemistry and Biochemistry (KU Leuven, Belgium) for assistance with the production of gluten hydrolysates. 2629
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(21) Lowry, O. H.; Rosebrough, N. J.; Farr, L. A.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193, 265−275. (22) Walstra, P. The roles of proteins and peptides in formation and stabilisation of emulsions. In Gums and Stabilisers for the Food Industry 11; Williams, P. A., Phillips, G. O., Eds.; Royal Society of Chemistry: London, UK, 2002; Vol. 11, pp 237−244. (23) Takeda, T.; Matsumura, Y.; Shimizu, M. Emulsifying and surface properties of wheat gluten under acidic conditions. J. Food Sci. 2001, 66, 393−399. (24) Ruiz-Henestrosa, V. P.; Sanchez, C. C.; Yust, M. d. M.; Pedroche, J.; Millan, F.; Patino, J. M. R. Limited enzymatic hydrolysis can improve the interfacial and foaming characteristics of βconglycinin. J. Agric. Food Chem. 2007, 55, 1536−1545. (25) Ma, H.; Forsell, P.; Partanen, R.; Seppanen, R.; Buchert, J.; Boer, H. Sodium caseinates with an altered isoelectric point as emulsifiers in oil/water systems. J. Agric. Food Chem. 2009, 57, 3800−3807. (26) Ma, H.; Forssell, P.; Partanen, R.; Buchert, J.; Boer, H. Charge modifications to improve the emulsifying properties of whey protein isolate. J. Agric. Food Chem. 2011, 59, 13246−13253. (27) Katsuda, M. S.; McClements, D. J.; Miglioranza, L. H. S.; Decker, E. A. Physical and oxidative stability of fish oil-in-water emulsions stabilized with β-lactoglobulin and pectin. J. Agric. Food Chem. 2008, 56, 5926−5931. (28) Chanamai, R.; McClements, D. J. Comparison of gum arabic, modified starch, and whey protein isolate as emulsifiers: influence of pH, CaCl2 and temperature. J. Food Sci. 2002, 67, 120−125. (29) Demetriades, K.; Coupland, J. N.; McClements, D. J. Physicochemical properties of whey protein-stabilized emulsions as affected by heating and ionic strength. J. Food Sci. 1997, 62, 462−467. (30) Tang, L.; Sun, J.; Zhang, H. C.; Zhang, C. S.; Yu, L. N.; Bi, J.; Zhu, F.; Liu, S. F.; Yang, Q. L. Evaluation of physicochemical and antioxidant properties of peanut protein hydrolysate. PLoS One 2012, 7, 1−7. (31) Ma, H.; Forssell, P.; Partanen, R.; Buchert, J.; Boer, H. Improving laccase catalyzed cross-linking of whey protein isolate and their application as emulsifiers. J. Agric. Food Chem. 2011, 59, 1406− 1414. (32) Onsaard, E.; Vittayanont, M.; Srigam, S.; McClements, D. J. Properties and stability of oil-in-water emulsions stabilized by coconut skim milk proteins. J. Agric. Food Chem. 2005, 53, 5747−5753. (33) Turgeon, S. L.; Gauthier, S. F.; Molle, D.; Leonil, J. Interfacial properties of tryptic peptides of β-lactoglobulin. J. Agric. Food Chem. 1992, 40, 669−675. (34) Thewissen, B. G.; Celus, I.; Brijs, K.; Delcour, J. A. Foaming properties of tryptic gliadin hydrolysate peptide fractions. Food Chem. 2011, 128, 606−612. (35) O’Meara, M. Determination of the interfacial tension between oilsteam and oil-air at elevated temperatures. North Carolina State University, Raleigh, NC, USA, 2012.
ABBREVIATIONS USED Alc, alcalase; DH, degree of hydrolysis; FITC, fluorescein isothiocyanate; GH, gluten hydrolysate; Try, trypsin
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REFERENCES
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dx.doi.org/10.1021/jf5001343 | J. Agric. Food Chem. 2014, 62, 2623−2630
