Patrick Giefer, Sabrina Bäther, Nadine Kaufmes, Helena Kieserling,
Anja Heyse, Wiebe Wagemans, Lars Barthel, Vera Meyer, Emanuel
Schneck, Udo Fritsching, Anja Maria Wagemans
Characterization of beta-lactoglobulin adsorption
on silica membrane pore surfaces and its impact
on membrane emulsification processes
Open Access via institutional repository of Technische Universität Berlin
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Journal article | Accepted version
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publication; also known as: Author’s Accepted Manuscript (AAM), Final Draft, Postprint)
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Citation details
Giefer, P., Bäther, S., Kaufmes, N., Kieserling, H., Heyse, A., Wagemans, W., Barthel, L., Meyer, V., Schneck,
E., Fritsching, U., & Wagemans, A. M. (2023). Characterization of -lactoglobulin adsorption on silica
membrane pore surfaces and its impact on membrane emulsification processes. In Journal of Colloid and
Interface Science (Vol. 652, pp. 1074–1084). Elsevier BV. https://doi.org/10.1016/j.jcis.2023.08.103.
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Characterization of β-lactoglobulin adsorption on silica membrane pore surfaces and its 1
impact on membrane emulsification processes 2
3
Patrick Giefer1,8+, Sabrina Bäther2+, Nadine Kaufmes2, Helena Kieserling2,3, Anja Heyse4, 4
Wiebe Wagemans5, Lars Barthel6, Vera Meyer6, Emanuel Schneck7, Udo Fritsching1,8, Anja 5
Maria Wagemans2*
6
+ These authors equally contributed to the manuscript 7
1: Leibniz Institute for Materials Engineering-IWT, Badgasteiner Straße 3, 28359 Bremen, 8
Germany 9
2: Technische Universität Berlin, Institute of Food Technology and Food Chemistry, 10
Department of Food Biosciences, Straße des 17. Juni 135, 10623 Berlin, Germany 11
3: Technische Universität Berlin, Institute of Food Technology and Food Chemistry, 12
Department of Food Chemistry and Analysis, Straße des 17. Juni 135, 10623 Berlin, 13
Germany 14
4: Technische Universität Berlin, Institute of Food Technology and Food Chemistry, 15
Department of Food Technology and Food Material Science, Straße des 17. Juni 135, 10623 16
Berlin, Germany 17
5: Fehrbelliner Straße 52a, 10119 Berlin, Germany 18
6: Technische Universität Berlin, Institute of Biotechnology, Department of Applied and 19
Molecular Microbiology, Straße des 17. Juni 135, 10623 Berlin, Germany 20
7: Technical University of Darmstadt, Department of Physics, 64277 Darmstadt, Germany 21
8: University of Bremen, Particles and Process Engineering, Bibliothekstraße 1, 28359 Bremen, 22
Germany 23
24
* Author of correspondence: 25
Anja Maria Wagemans 26
Department of Food Biosciences 27
Technische Universität Berlin 28
Königin-Luise-Str. 22, 14195 Berlin, Germany 29
Tel.: 0049-30-31471833; 30
Mail: wagemans@tu-berlin.de 31
ABSTRACT 32
Protein adsorption plays a key role in membrane fouling in liquid processing, but the specific 33
underlying molecular mechanisms of β-lactoglobulin adsorption on ceramic silica surfaces in 34
premix membrane emulsification have not been investigated yet. 35
In this study, we aimed to elucidate the β-lactoglobulin adsorption and its effect on the premix 36
membrane emulsification of β-lactoglobulin-stabilized oil-in-water emulsions. In particular, the 37
conformation, molecular interactions, layer thickness, surface energy of the adsorbed β-38
lactoglobulin and resulting droplet size distribution are investigated in relation to the solvent 39
properties (aggregation state of β-lactoglobulin) and the treatment of the silica surface 40
(hydrophilization). 41
The β-lactoglobulin adsorption is driven by attractive electrostatic interactions between 42
positively charged amino acid residues, i.e., lysin and negatively charged silanol groups, and is 43
stabilized by hydrophobic interactions. The strong negative charges of the treated silica surfaces 44
result in a high apparent layer thickness of β-lactoglobulin. Although the conformation of the 45
adsorbed β-lactoglobulin layer varies with membrane treatment and the solvent properties, the 46
β-lactoglobulin adsorption offsets the effect of hydrophilization of the membrane so that the 47
surface energies after β-lactoglobulin adsorption are comparable. The resulting droplet size 48
distribution of oil-in-water emulsions produced by premix membrane emulsification are similar 49
for treated and untreated silica surfaces. 50
KEY WORDS 51
Protein adsorption, conformation, molecular interactions, fouling, ceramic membranes 52
1 INTRODUCTION 53
Premix membrane emulsification (PME) is an emulsification technique for the preparation of 54
emulsions of sensitive biomaterials, such as oil-in-water (O/W) emulsions, which are relevant 55
in several industries. In industries such as food, pharmaceuticals, cosmetics and chemicals, 56
PME is used for the preparation of specific emulsions for consumer and technical products [1], 57
for the encapsulation and delivery of bioactive components [2], and for the synthesis of 58
micro−/nanomaterials [3]. In this technique, a coarse premix is forced through a porous 59
membrane by a pressure gradient. Glass membranes, such as borosilicate membranes, are often 60
used in PME, because the membrane properties – such as pore size, surface roughness and 61
polarity – can be easily modified [4–7]. Borosilicate membranes can be used to produce fine 62
emulsions with a narrow droplet size distribution [8]. Droplet size can be controlled by the 63
transmembrane pressure, pore size and number of cycles [9]. In addition, the droplet size can 64
also be influenced by the membrane surface properties (wettability), as the shear deformation 65
of oil droplets in the emulsion is influenced by the interactions between the oil phase, water 66
phase and membrane material, at the so-called three-phase contact line [7,10]. In addition, the 67
type of emulsifier also has a significant effect on the emulsion produced. High molecular weight 68
emulsifiers such as proteins are often preferred to over synthetic low molecular weight 69
surfactants such as monoglycerides [11]. 70
The stabilization process of O/W interfaces through proteins is complex , but can be described 71
as a three-stage process [12]: In stage (I), the protein migrates through the bulk water phase, in 72
stage (II), the protein adsorbs at the oil/water-interface, and in stage (III), the protein forms an 73
interfacial protein film. A well described model protein is the whey protein β-lactoglobulin (β-74
lg). In previous studies, we investigated the changes of the molecular interactions and the 75
interfacial properties of β-lg as a function of the β-lg aggregation state, which we modified 76
through the solvent properties, specifically using pH 7, pH 7 with 100 mM NaCl and pH 9 (pH 77
7, pH 7NaCl and pH 9) [13–17]. For pH values close to 7, an equilibrium of the monomeric and 78
dimeric forms could be observed [18]. Higher ionic strengths cause a screening of electrostatic 79
charges, shifting the equilibrium to the dimer side [18–20]. At higher pH values, deprotonation 80
leads to negatively charged β-lg, and, due to electrostatic repulsion, the monomeric form 81
dominates [21,22]. Because the solvent properties influence the sensitive monomer-dimer 82
equilibrium, they might also affect the β-lg adsorption leading to a mono- or multi β-lg layers 83
at the O/W interface [4]. This in turn, affects the stabilization process and the droplet size 84
distribution of the emulsion. 85
Besides adsorbing at the O/W interface, β-lg also adsorbs on the membrane surfaces within the 86
PME, which potentially leads to clogging of the pores and the formation of a layer on the 87
membrane surface. This, so-called, fouling of the membrane limits continuous processing and 88
upscaling for industrial applications [23,24]. Moreover, it can lead to a loss of β-lg to function 89
as emulsifier [25, 26]. Fouling is defined as a decrease in membrane performance due to an 90
increasing resistance over time, leading to s an undesired decrease in flux at constant 91
transmembrane pressure [27]. As consequence, membranes need to be regenerated through 92
cleaning procedures or need to be replaced, both lowering the overall efficiency [28]. 93
Fouling is determined by the affinity of the β-lg to the membrane. For adsorption of β-lg to 94
silica surfaces, relevant molecular interactions are electrostatic interactions, hydrogen bonds, 95
hydrophobic interactions as well as van der Waals interactions. For silicas, the silanol groups 96
are deprotonated above the pKa (pH 7.1) [29] and, therefore, exhibit negative charges at neutral 97
pH values [30]. Likewise, β-lg is negatively charged above its isoelectric point, which is pH 5.2 98
- 5.3 [21,31]. Consequently, at neutral pH values electrostatic repulsion between β-lg and the 99
membrane is very likely. However, the presence of local electrostatic attraction between silanol 100
groups and amino acid residues of lysin are also possible. In general, electrostatic interactions 101
can be altered by ions, which is especially true for the β-lg sample pH 7NaCl. Furthermore, 102
hydrophobic interactions can be relevant, since β-lg has hydrophobic residues that can be 103
exposed due to unfolding, for instance at pH 9 [14]. The silica surfaces can also exhibit 104
hydrophobic patches due to organic contaminants or non-oxidized silicon atoms [32], 105
facilitating attractive hydrophobic interactions between β-lg and the membrane. Additionally, 106
van der Waals forces lead to attractive interactions supporting the adsorption. The 107
hydrophilicity of the membrane can be increased through a treatment with piranha, which has 108
two effects. Firstly, it removes the organic contaminants and consequently decreases the 109
hydrophobic interactions [32, 33]. Secondly, the silicon atoms are oxidized and silanol groups 110
are formed, leading to an increase in the hydrophilicity and negative charges of the treated 111
membranes [30]. This in turn affects the molecular interactions between β-lg and the membrane, 112
influencing adsorption, the wetting behavior and consequently the oil droplet size distribution 113
in the O/W emulsions. 114
Although fouling is a well-described phenomenon in membrane-related processes, the 115
molecular interactions responsible for the adsorption of β-lg on silica surfaces have not been 116
discussed in detail in the literature. In particular, the adsorption of β-lg on silica surfaces as 117
function of the aggregation state has not been extensively investigated. Only, Elofsson et al. 118
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