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Properties of #-lactoglobulin aggregates and gels as affected by ternary emulsifier mixtures of Tween 20, lecithin and sucrose palmitate Verena Wiedenmann, Michaela Frister, Kathleen Oehlke, Ulrike van der Schaaf, and Heike Karbstein J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02480 • Publication Date (Web): 23 Jul 2019 Downloaded from pubs.acs.org on July 23, 2019
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Properties of β-lactoglobulin aggregates and gels as affected by ternary emulsifier mixtures of Tween 20, lecithin and sucrose palmitate
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Verena Wiedenmann1,2, Michaela Frister, Kathleen Oehlke1, Ulrike van der Schaaf², Heike Petra Karbstein²
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1Max
Rubner-Institut, Federal Research Institute of Nutrition and Food, Department of Food Technology and Bioprocess Engineering, Karlsruhe, Germany ²Karlsruhe Institute of Technology, Institute of Process Engineering in Life Sciences, Chair for Food Process Engineering, Karlsruhe, Germany
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Keywords: protein aggregates, texture, surfactant-protein interactions, aggregate size
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Abstract
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The influence of sucrose palmitate, Tween 20 and lecithin on the properties of heat-induced
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aggregates and cold set gels of β-lactoglobulin was studied based on an experimental
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mixture design with a fixed total emulsifier concentration. Emulsifiers were added to the
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protein solution before heating. Aggregate size and absolute values of zeta potential
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increased with the addition of emulsifiers, among which lecithin had the most pronounced
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effect. The water retention of the aggregates correlated positively with the aggregate size.
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Gels had reduced fracture stress and strains with increasing sucrose palmitate and
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decreasing Tween 20 contents. The fracture properties correlated with the zeta potentials of
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the aggregates and larger aggregates led to gels with higher water holding capacities.
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The emulsifiers hence influenced the gel properties indirectly via the aggregate properties.
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The impact of emulsifiers on food structures should therefore be considered when a food
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product is designed.
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1. Introduction
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Low molecule weight emulsifiers are important in the food sector: Their applications include
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stabilizing oil droplets in emulsions like mayonnaise, controlling fat agglomeration or
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coalescence for example in whipped cream and modifying the viscosity in chocolate melts 1.
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Besides fulfilling their primary function, the emulsifiers interact with the surrounding food
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components. For example, it is well known that low molecule weight emulsifiers can interact
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with globular proteins which are present in many foods like dairy products 2.
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β-lactoglobulin (BLG) is the major component of whey protein and tends to dominate its
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techno-functional properties. BLG consists of 162 amino acid residues and has two disulfide
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bonds and one free thiol group. This thiol group is critical in the formation of heat induced
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protein aggregates by intermolecular thiol/disulfide exchange reactions. At pH 7, BLG has
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several binding sites for small molecules like fatty acids, retinoids, and surfactants
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interactions between emulsifiers and BLG are of great importance e.g. in food emulsions and
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therefore gained much attention within the last years
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found to cause unfolding of the protein structure, whereas non-ionic emulsifiers did not
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denature or destabilize proteins at room temperature 10.
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During food processing, proteins are often subjected to heat treatment. BLG is known to
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unfold irreversibly and to aggregate at temperatures above 75 to 80 °C. During heating, the
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protein unfolds and forms small oligomers. At higher protein concentrations, these oligomers
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associate into aggregates, which is dominated by disulfide bridges and hydrophobic forces.
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However, also van der Waals and ionic interactions are involved in the aggregation
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process11-12. The ionic strength and the pH value of the protein solution as well as protein
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concentration significantly affect aggregate properties such as size, shape and density
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Aggregates are known to become larger at high ionic strength and at pH-values close to the
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isoelectric point due to reduced isoelectric repulsion
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aggregates have a spherical, rod-like or worm-like shape
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revealed that BLG aggregate properties were modified by emulsifiers that were bound to the
6-9.
3-6.
The
Especially ionic emulsifiers were
15.
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Depending on the conditions, 13, 16.
Previous studies also
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protein: Anionic and nonionic emulsifiers reduced the protein-protein association by
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solubilizing the proteins and thus reduced the aggregate sizes. The required surfactant
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concentration depended on the nature of the surfactant and its properties. The non-ionic
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surfactant alkyl maltopyranosides only affected aggregation of BLG at concentrations above
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its critical micelle concentration (cmc)
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bound to non-aggregated BLG far below its cmc leading to a reduced heat-induced
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aggregation rate of the protein and smaller aggregates
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size of aggregates at pH-values above the isoelectric point due to reduction of the protein net
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charge 10.
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Complex food systems often contain a combination of different emulsifiers. To our
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knowledge, the impact of a mixture of different emulsifiers on heat induced food protein
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aggregation has not yet been studied. Tween 20, sucrose palmitate and lecithin can be used
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to stabilize solid lipid nanoparticles
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likely relevant to the formation of a food structure. Tween 20, e.g., exhibits a cloud point
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sucrose palmitate tends to form gels
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study aimed to investigate how these different emulsifiers and a combination thereof
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influence the properties of protein aggregates. Such information would be especially
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important, as protein aggregates are precursors for gels. Properties of protein aggregates
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and emulsifiers are known to influence the properties of protein gels
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study, we observed that emulsifiers influenced the properties of heat-set protein gels that
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were formed in the presence of emulsifier stabilized solid lipid nanoparticles
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knowledge about the effect of multiple emulsifier mixtures on protein aggregates and gels is
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crucial for the targeted design of food products. This paper investigates the impact of the
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three emulsifiers Tween 20, sucrose palmitate (SP) and lecithin in different combinations on
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the properties of heat-induced aggregation of BLG.
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The aggregates were formed in the absence and presence of the emulsifiers and were
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characterized regarding their size, zeta potential, water holding, and viscosity. To investigate
18.
20
10.
By contrast, the anionic sodium dodecyl sulfate
13, 17.
Cationic ligands increased the
These emulsifiers have different properties that are 19,
and lecithin has a zwitter-ionic nature. The present
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In our previous
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Hence,
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how these aggregate properties influenced the later gel properties, we chose five emulsifier
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mixtures that resulted in a broad range of aggregate sizes and water retention. Cold set BLG
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gels were formed with these protein aggregates and the gels were characterized regarding
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their mechanical properties and water holding capacity.
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2. Materials and methods
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2.1. Materials
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BiPro whey protein isolate was kindly donated by Agropur Ingredients (Eden Prairie, MN,
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USA). Tween 20® (Polyoxyethylene sorbitan monolaurate, Tween 20) was purchased from
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Sigma Aldrich (St. Louis, Missouri, USA), glucono-δ-lactone (GDL) and sucrose palmitate
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from Alfa Aesar (Karlsruhe, Germany). Soy lecithin (Emulpur IP) was kindly donated by
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Cargill
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phosphate was purchased from Carl Roth GmbH (Karlsruhe, Germany), hydrochloric acid,
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sodium hydroxide and di-Sodium hydrogen phosphate from Merck KGaA (Darmstadt,
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Germany). All solutions were prepared in demineralized water.
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(Cargill
Texturizing
Solutions,
Hamburg,
Germany).
Potassium-di-hydrogen
2.2. Purification of β-lactoglobulin (BLG)
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BLG was isolated from whey protein isolate (WPI), following a method described by Keppler
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et al.
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water and hydrated for 18 h at 8 °C. Subsequently, the pH value was adjusted to 4.8 with
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hydrochloric acid to precipitate remaining caseins. Caseins were separated by centrifugation
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at 3220 g for 20 min. The pH value of the remaining protein solution was then set to 3.8 with
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hydrochloric acid and heated to 55 °C for 30 min. During this heat treatment, all whey
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proteins except BLG precipitated and were removed by centrifugation at 20 °C at 3220 g for
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20 min. The pH value of the remaining supernatant was readjusted to 7.0 with sodium
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hydroxide before washing the protein three times with ultrapure water by ultrafiltration
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(Amicon Ultra-15, PLGC Ultracel-PL Membran MWCO of 10 kDa, Merck KGaA, Darmstadt,
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Germany). BLG solution was collected, freeze dried and kept at room temperature until use.
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with slight modifications: In short, 20% (w/w) WPI was dissolved in demineralized
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The denaturation degree of BLG was determined using the respective German Industrial
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Standard procedure (DIN 10473, German Industrial Standard, 1997). Samples were
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analyzed before and after a pH-adjustment to 4.6 with hydrochloric acid. At this pH,
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denatured BLG would precipitate and could be removed by filtration (syringe filter, 0.2 µm,
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Merck KGaA, Darmstadt, Germany). The concentrations and purity of the supernatants were
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determined using reversed phase-high performance liquid chromatography (Agilent 1290
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Infinity LC System HPLC) with a fluorescence detector and C-18 reversed-phase column
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(AerisTM XB-C18 Wide Pore 3.6 µm, 200 Å LC Column 50 x 2.1 mm, Phenomenex,
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Torrance, United States). The injection volume was set to 10 μL at a flow rate of
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1.2 mL·min−1 and a column temperature of 40 °C. Eluents A (0.1% (v/v) trifluoroacetic acid
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(TFA) in water) and B (0.1% TFA (v/v) in acetonitrile) were used. Used elution gradient steps
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were 35 – 42.5% B (1–12.5 min), 42.5 – 46% B (12.5 – 20.5 min), 46 – 35% B (20.5 –
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22 min), and 35% B (22 – 23 min). Fluorescence was monitored at excitation and emission
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wavelengths of 225 and 340 nm, respectively. The degree of denaturation corresponded to
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the relative difference of the BLG concentration before and after precipitation and was below
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1%.
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2.3. Preparation of emulsifier-protein mixtures and heat treatment
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In order to model the influence of sucrose palmitate, Tween 20 and soy lecithin on the
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properties of BLG aggregates, an experimental design method was used (Figure 1). The total
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emulsifier concentration was kept at 60 mM as this emulsifier concentration is typically used
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for production of solid lipid nanoparticles
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figures within this work, the emulsifier concentrations are indicated as percent of 60 mM. As
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lecithin is only slightly water soluble, its content was limited to a maximum of 25% (equals
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15 mM). The experimental design was calculated applying JMP (14.3.0, SAS institutes). The
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central point of the matrix as well as the corners were repeated four times. All other samples
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were triplicates, duplicates or performed once, according to the experimental design. The
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60 mM was set as 100%, and in all ternary
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exact design including sample identification and block numbers are given in the
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supplemental data.
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Emulsifier solutions were prepared by dissolving the respective amount of emulsifier in 5 mM
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phosphate buffer solution at a pH value of 7.0. Dissolution was accelerated by heating to
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50 °C and if necessary, sonication in an ultrasound water bath for 1 h. The emulsifier mixture
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of 7.5% (4.5 mM) lecithin and 93.5% (55.5 mM) SP could not be dissolved. Thus, values for
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aggregates prepared with this emulsifier composition were not determined.
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BLG powder (5.6% (w/w)) was dissolved in buffer (control) or emulsifier solution. The
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solution was left to hydrate for at least 18 h at 8 °C. The aggregation of the protein was
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achieved by heat treatment under stirring at 90 °C for 30 min. After heat treatment, the
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samples were immediately cooled to 20 ± 1 °C in ice water.
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2.4. Determination of aggregate size and zeta potential
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Particle size and zeta potential were analyzed using a ZetaSizer Nano ZS (Malvern
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Instruments, UK). Zeta potentials were measured via electrophoretic mobility. Prior to the
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measurements, the sample conductivity was set to 50 µS/cm by diluting with ultrapure water.
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The aggregate sizes of the samples were determined by dynamic light scattering with a
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backscattering angle of 173°. The z-averages were analyzed based on the intensity based
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mean diameters (z-average) using Mie theory. Values for z-averages were considered
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satisfactory if the polydispersity index was
