Subscriber access provided by Nottingham Trent University
Food and Beverage Chemistry/Biochemistry
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 34
Journal of Agricultural and Food Chemistry
1 2
Properties of β-lactoglobulin aggregates and gels as affected by ternary emulsifier mixtures of Tween 20, lecithin and sucrose palmitate
3 4
Verena Wiedenmann1,2, Michaela Frister, Kathleen Oehlke1, Ulrike van der Schaaf², Heike Petra Karbstein²
5 6 7 8 9
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
10
Keywords: protein aggregates, texture, surfactant-protein interactions, aggregate size
11
Abstract
12
The influence of sucrose palmitate, Tween 20 and lecithin on the properties of heat-induced
13
aggregates and cold set gels of β-lactoglobulin was studied based on an experimental
14
mixture design with a fixed total emulsifier concentration. Emulsifiers were added to the
15
protein solution before heating. Aggregate size and absolute values of zeta potential
16
increased with the addition of emulsifiers, among which lecithin had the most pronounced
17
effect. The water retention of the aggregates correlated positively with the aggregate size.
18
Gels had reduced fracture stress and strains with increasing sucrose palmitate and
19
decreasing Tween 20 contents. The fracture properties correlated with the zeta potentials of
20
the aggregates and larger aggregates led to gels with higher water holding capacities.
21
The emulsifiers hence influenced the gel properties indirectly via the aggregate properties.
22
The impact of emulsifiers on food structures should therefore be considered when a food
23
product is designed.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 2 of 34
24
1. Introduction
25
Low molecule weight emulsifiers are important in the food sector: Their applications include
26
stabilizing oil droplets in emulsions like mayonnaise, controlling fat agglomeration or
27
coalescence for example in whipped cream and modifying the viscosity in chocolate melts 1.
28
Besides fulfilling their primary function, the emulsifiers interact with the surrounding food
29
components. For example, it is well known that low molecule weight emulsifiers can interact
30
with globular proteins which are present in many foods like dairy products 2.
31
β-lactoglobulin (BLG) is the major component of whey protein and tends to dominate its
32
techno-functional properties. BLG consists of 162 amino acid residues and has two disulfide
33
bonds and one free thiol group. This thiol group is critical in the formation of heat induced
34
protein aggregates by intermolecular thiol/disulfide exchange reactions. At pH 7, BLG has
35
several binding sites for small molecules like fatty acids, retinoids, and surfactants
36
interactions between emulsifiers and BLG are of great importance e.g. in food emulsions and
37
therefore gained much attention within the last years
38
found to cause unfolding of the protein structure, whereas non-ionic emulsifiers did not
39
denature or destabilize proteins at room temperature 10.
40
During food processing, proteins are often subjected to heat treatment. BLG is known to
41
unfold irreversibly and to aggregate at temperatures above 75 to 80 °C. During heating, the
42
protein unfolds and forms small oligomers. At higher protein concentrations, these oligomers
43
associate into aggregates, which is dominated by disulfide bridges and hydrophobic forces.
44
However, also van der Waals and ionic interactions are involved in the aggregation
45
process11-12. The ionic strength and the pH value of the protein solution as well as protein
46
concentration significantly affect aggregate properties such as size, shape and density
47
Aggregates are known to become larger at high ionic strength and at pH-values close to the
48
isoelectric point due to reduced isoelectric repulsion
49
aggregates have a spherical, rod-like or worm-like shape
50
revealed that BLG aggregate properties were modified by emulsifiers that were bound to the
6-9.
3-6.
The
Especially ionic emulsifiers were
15.
ACS Paragon Plus Environment
13-14:
Depending on the conditions, 13, 16.
Previous studies also
Page 3 of 34
Journal of Agricultural and Food Chemistry
51
protein: Anionic and nonionic emulsifiers reduced the protein-protein association by
52
solubilizing the proteins and thus reduced the aggregate sizes. The required surfactant
53
concentration depended on the nature of the surfactant and its properties. The non-ionic
54
surfactant alkyl maltopyranosides only affected aggregation of BLG at concentrations above
55
its critical micelle concentration (cmc)
56
bound to non-aggregated BLG far below its cmc leading to a reduced heat-induced
57
aggregation rate of the protein and smaller aggregates
58
size of aggregates at pH-values above the isoelectric point due to reduction of the protein net
59
charge 10.
60
Complex food systems often contain a combination of different emulsifiers. To our
61
knowledge, the impact of a mixture of different emulsifiers on heat induced food protein
62
aggregation has not yet been studied. Tween 20, sucrose palmitate and lecithin can be used
63
to stabilize solid lipid nanoparticles
64
likely relevant to the formation of a food structure. Tween 20, e.g., exhibits a cloud point
65
sucrose palmitate tends to form gels
66
study aimed to investigate how these different emulsifiers and a combination thereof
67
influence the properties of protein aggregates. Such information would be especially
68
important, as protein aggregates are precursors for gels. Properties of protein aggregates
69
and emulsifiers are known to influence the properties of protein gels
70
study, we observed that emulsifiers influenced the properties of heat-set protein gels that
71
were formed in the presence of emulsifier stabilized solid lipid nanoparticles
72
knowledge about the effect of multiple emulsifier mixtures on protein aggregates and gels is
73
crucial for the targeted design of food products. This paper investigates the impact of the
74
three emulsifiers Tween 20, sucrose palmitate (SP) and lecithin in different combinations on
75
the properties of heat-induced aggregation of BLG.
76
The aggregates were formed in the absence and presence of the emulsifiers and were
77
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
ACS Paragon Plus Environment
21-24.
In our previous
25.
Hence,
Journal of Agricultural and Food Chemistry
Page 4 of 34
78
how these aggregate properties influenced the later gel properties, we chose five emulsifier
79
mixtures that resulted in a broad range of aggregate sizes and water retention. Cold set BLG
80
gels were formed with these protein aggregates and the gels were characterized regarding
81
their mechanical properties and water holding capacity.
82
2. Materials and methods
83
2.1. Materials
84
BiPro whey protein isolate was kindly donated by Agropur Ingredients (Eden Prairie, MN,
85
USA). Tween 20® (Polyoxyethylene sorbitan monolaurate, Tween 20) was purchased from
86
Sigma Aldrich (St. Louis, Missouri, USA), glucono-δ-lactone (GDL) and sucrose palmitate
87
from Alfa Aesar (Karlsruhe, Germany). Soy lecithin (Emulpur IP) was kindly donated by
88
Cargill
89
phosphate was purchased from Carl Roth GmbH (Karlsruhe, Germany), hydrochloric acid,
90
sodium hydroxide and di-Sodium hydrogen phosphate from Merck KGaA (Darmstadt,
91
Germany). All solutions were prepared in demineralized water.
92
(Cargill
Texturizing
Solutions,
Hamburg,
Germany).
Potassium-di-hydrogen
2.2. Purification of β-lactoglobulin (BLG)
93
BLG was isolated from whey protein isolate (WPI), following a method described by Keppler
94
et al.
95
water and hydrated for 18 h at 8 °C. Subsequently, the pH value was adjusted to 4.8 with
96
hydrochloric acid to precipitate remaining caseins. Caseins were separated by centrifugation
97
at 3220 g for 20 min. The pH value of the remaining protein solution was then set to 3.8 with
98
hydrochloric acid and heated to 55 °C for 30 min. During this heat treatment, all whey
99
proteins except BLG precipitated and were removed by centrifugation at 20 °C at 3220 g for
100
20 min. The pH value of the remaining supernatant was readjusted to 7.0 with sodium
101
hydroxide before washing the protein three times with ultrapure water by ultrafiltration
102
(Amicon Ultra-15, PLGC Ultracel-PL Membran MWCO of 10 kDa, Merck KGaA, Darmstadt,
103
Germany). BLG solution was collected, freeze dried and kept at room temperature until use.
9
with slight modifications: In short, 20% (w/w) WPI was dissolved in demineralized
ACS Paragon Plus Environment
Page 5 of 34
Journal of Agricultural and Food Chemistry
104
The denaturation degree of BLG was determined using the respective German Industrial
105
Standard procedure (DIN 10473, German Industrial Standard, 1997). Samples were
106
analyzed before and after a pH-adjustment to 4.6 with hydrochloric acid. At this pH,
107
denatured BLG would precipitate and could be removed by filtration (syringe filter, 0.2 µm,
108
Merck KGaA, Darmstadt, Germany). The concentrations and purity of the supernatants were
109
determined using reversed phase-high performance liquid chromatography (Agilent 1290
110
Infinity LC System HPLC) with a fluorescence detector and C-18 reversed-phase column
111
(AerisTM XB-C18 Wide Pore 3.6 µm, 200 Å LC Column 50 x 2.1 mm, Phenomenex,
112
Torrance, United States). The injection volume was set to 10 μL at a flow rate of
113
1.2 mL·min−1 and a column temperature of 40 °C. Eluents A (0.1% (v/v) trifluoroacetic acid
114
(TFA) in water) and B (0.1% TFA (v/v) in acetonitrile) were used. Used elution gradient steps
115
were 35 – 42.5% B (1–12.5 min), 42.5 – 46% B (12.5 – 20.5 min), 46 – 35% B (20.5 –
116
22 min), and 35% B (22 – 23 min). Fluorescence was monitored at excitation and emission
117
wavelengths of 225 and 340 nm, respectively. The degree of denaturation corresponded to
118
the relative difference of the BLG concentration before and after precipitation and was below
119
1%.
120
2.3. Preparation of emulsifier-protein mixtures and heat treatment
121
In order to model the influence of sucrose palmitate, Tween 20 and soy lecithin on the
122
properties of BLG aggregates, an experimental design method was used (Figure 1). The total
123
emulsifier concentration was kept at 60 mM as this emulsifier concentration is typically used
124
for production of solid lipid nanoparticles
125
figures within this work, the emulsifier concentrations are indicated as percent of 60 mM. As
126
lecithin is only slightly water soluble, its content was limited to a maximum of 25% (equals
127
15 mM). The experimental design was calculated applying JMP (14.3.0, SAS institutes). The
128
central point of the matrix as well as the corners were repeated four times. All other samples
129
were triplicates, duplicates or performed once, according to the experimental design. The
18.
60 mM was set as 100%, and in all ternary
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
130
exact design including sample identification and block numbers are given in the
131
supplemental data.
132
Emulsifier solutions were prepared by dissolving the respective amount of emulsifier in 5 mM
133
phosphate buffer solution at a pH value of 7.0. Dissolution was accelerated by heating to
134
50 °C and if necessary, sonication in an ultrasound water bath for 1 h. The emulsifier mixture
135
of 7.5% (4.5 mM) lecithin and 93.5% (55.5 mM) SP could not be dissolved. Thus, values for
136
aggregates prepared with this emulsifier composition were not determined.
137
BLG powder (5.6% (w/w)) was dissolved in buffer (control) or emulsifier solution. The
138
solution was left to hydrate for at least 18 h at 8 °C. The aggregation of the protein was
139
achieved by heat treatment under stirring at 90 °C for 30 min. After heat treatment, the
140
samples were immediately cooled to 20 ± 1 °C in ice water.
141
2.4. Determination of aggregate size and zeta potential
142
Particle size and zeta potential were analyzed using a ZetaSizer Nano ZS (Malvern
143
Instruments, UK). Zeta potentials were measured via electrophoretic mobility. Prior to the
144
measurements, the sample conductivity was set to 50 µS/cm by diluting with ultrapure water.
145
The aggregate sizes of the samples were determined by dynamic light scattering with a
146
backscattering angle of 173°. The z-averages were analyzed based on the intensity based
147
mean diameters (z-average) using Mie theory. Values for z-averages were considered
148
satisfactory if the polydispersity index was