of O il/Water-Interfaces
vorgelegt von
Diplom Lebensmittelchemikerin Helena Kieserling
ORCID: 0000-0002-8093-6794
an der Fakultät III – Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften – Dr. rer. nat. –
genehmigte Dissertation
Promotionsausschuss:
Vorsitzender: Prof. Dr. Hajo Haase Gutachter: Prof. Dr. Stephan Drusch
Gutachterin: Prof. Dr. Anja Maria Wagemans Gutachter: Prof. Dr. Matteo Mario Scampicchio Tag der wissenschaftlichen Aussprache: 29. Januar 2021
Berlin 2021
First of all, I would like to thank my supervisor Prof. Dr. Stephan Drusch for his guidance during probably one of the most formative periods of my life. Thank you for the opportunity to learn about the many facets of science, including experimental lab work, interpretation of results and writing texts of any kind. You have taken each of my texts to a new level, allowing me to see beyond my own limitations, because probably no one can structure as well as you can, and no one finds the red line better than you do. Prof. Stephan Drusch has, thus, taught me what quality means.
I want to thank Prof. Dr. Anja Maria Wagemans, my supervisor and mentor. Nobody is as motivating as you are. You saw the best in me and never shied away from supporting me, no matter in which situation. Thank you for all the optimism, the deep scientific discussions, and for allowing me to grow so much under your care. Prof. Dr. Anja Maria Wagemans, you taught me how to walk confidently through the scientific world.
I gratefully thank Prof. Dr. Matteo Scampicchio for his flexibility and the willingness to evaluate this thesis.
I appreciate the support of the Deutsche Forschungsgemeinschaft (DFG). This work was part of the priority project SPP 1934 “DispBiotech”. Thank you for the opportunity to build such a broad network full of brilliant scientists.
Special thanks go to my students, Katrin Rochberg as you were the very first student I was allowed to supervise, Annika Pankow for the standards you have set, Nadine Kaufmes for your attention to detail, Thu Nguyen and Alex Westphal for your relaxed and stringent way of working through things, Sarah Winkler for your perseverance and especially Ingalisa Alsmeier for the many years of great cooperation and the incredible support at all times.
Moreover, I would like to thank Julia Wolff, Thu Nguyen, Alex Westphal, Hannes Schäfer and Ingalisa Alsmeier for your support as tutors.
I would like to thank my colleagues at the Department of Food Technology and Material Science and especially the Department Food Colloids. Sabrina, Theresia, Marina, Artwin, Hanna, Mrs. Kern, Silvia, Sarah, Vivienne, Talita, Thanh, Mr. Blochwitz, Mrs. Ludewig, Alina, dear colleagues, thank you for all your support. Mrs. Stoll, it was a pleasure to give lessons together. Dagmar, I thank you for your relaxed attitude, no matter which form was filled out wrong. Martina and Monika, thank you for being there right in the next office and for always lending an ear to me. Christoph, my former neighbor, thank you for our rare but pleasant meetings in the corner bar. Sabrina, you were the best roommate I could have imagined, thank you for all the candy that made our time together sweeter. Anja, thank you for the relaxing talks about all our plans and for bringing Lipase into our project, I am looking forward to continuing to go through life together. Rocio, my favorite PostDoc, thank you for the 24/7 Drama-Beratung, for being there for each other. No matter the mood, in which our meetings started, you always managed to cheer me up, coming out of them laughi n onfident Sta s nduri arm-hearte s re!
Tobias and Patrick, thank you for the enriching molecular dynamic simulations. Vanessa and Maximilian, it was fun working with you, all collaborations should be this successful.
Jacqueline and Timon, I am so glad the SPP brought us together. Julia, thank you, you are my role model with whom I enjoyed discussing so much.
Thank you also for the fact that I can now call many colleagues friends.
Also, I would like to thank Sandra, Hendrik, Lisa, Jasmin and especially Max for accompanying me from afar. Ricarda, thank you for everything, I am waiting for our plans with the horses in our backyards. Xenia, I would have loved to walk the dissertation path together with you, but even from distance, across all the thousands of miles, we managed quite well. Fabi, thank you, you are the best and always in my heart. Thank you, Annette, now I know that you would really walk with me through hell, I am looking forward for all the challenges that we will manage side by side in the future.
Bärbel and Manni, Alex and Leni, I thank you for having welcomed me into your family, I could not imagine more caring people than you are. I thank my father for accompanying me in his own way. I thank my mother for teaching me how enriching education is and that one can only get far with hard work, and by teaching me this for bringing me to where I am now. I want to thank my sister, her husband and her kids, for our wonderful and cosy time together and the unlimited support – you are my basis!
The most beautiful thing my thesis has brought me is you, my beloved husband. Thank you for supporting me in every imaginable way. Without your bowls of fruit, candy, and crisps, I would not have made it through the last part of the thesis. We are a strong team and the fact that we can still say that after this phase in our lives, makes me look forward to our future with joy.
Food products based on oil/water-emulsions, such as beverages, dairy products, or targeted delivery systems for functional food ingredients, are thermodynamically unstable systems.
Over time, on a macro-level, the dispersed oil droplets in the water phase tend to flocculate, coalesce, and cream. The kinetics of these destabilization mechanisms depend on the emulsifier properties on the micro-level. During interfacial stabilization, emulsifiers such as whey protein -lactoglobulin ( -lg) form a protein film at the oil/water-interface, whose stability against mechanical stress contributes to the emulsion stability. The protein film stability, in turn, depends on molecular interactions of the adjacent proteins that are determined by the protein structure in the bulk water phase. However, so far only few studies described the protein structure at the oil/water interface, and none of them systematically connected the interfacial film stability with the protein structure during the interfacial stabilization process.
To understand and control the protein film stability at the oil/water-interface, this thesis aims to systematically investigate the influence of the protein structure on the (I) migration through the bulk water phase, (II) adsorption at the oil/water-interface and (III) formation of an interfacial protein film. For this purpose, the structure of the model protein -lg in the bulk water phase was intentionally modified by changing ambient conditions (pH, ionic strength) and by employing high hydrostatic pressure. Comprehensive experimental methodology allowed the characterization of the net charge, degree of unfolding, surface hydrophobicity, and molecular weight. The structural changes that -lg undergoes through adsorption at the oil/water interface were studied systematically. For this purpose, an in situ FTIR method was established to analyze the unfolding of proteins at the oil/water interface.
Subsequently, the protein structure was linked to the interfacial behavior and the film stability against lateral compression and expansion, shear, and dilatational deformation.
In summary, a slow (I) migration due to the combination of dimeric conformation and electrostatic repulsion reduced the protein film stability at high dilatational deformation, as a consequence of a reduced stabilization of the unoccupied oil/water-interface. The formation of pronounced intermolecular interactions during (III) protein film formation increased the stability against lateral compression and expansion, shear, and small dilatational deformation. Pronounced unfolding at the oil/water-interface and a high surface hydrophobicity enhanced intermolecular interactions and resulted in the highest protein film stability regardless of the net charge of the proteins. A strong reduction in interfacial tension during (II) adsorption at the oil/water-interface, as indicated by the highest adsorption rate, was not accompanied by a high film stability, since the electrostatically shielded proteins were not able to form a strong interfacial network due to minor interactions.
Finally, the findings deepen the understanding of the protein film stability at oil/water- interfaces by combining fundamental insights about the protein structure with interfacial stabilization mechanisms. For future analysis, this thesis presents a promising approach for the identification of new protein structures that result in stable interfacial films applicable
Lebensmittel, die aus Öl/Wasser-Emulsionen bestehen oder diese enthalten, sind thermodynamisch instabile Systeme. Auf der Makroebene neigen die dispergierten Öltröpfchen in der wässrigen Phase dazu, mit der Zeit auszuflocken, zu koaleszieren und aufzurahmen. Die Kinetik dieser Destabilisierungsmechanismen hängt auf der Mikroebene von den Eigenschaften des eingesetzten Emulgators ab. Bei der Grenzflächenstabilisierung bilden Emulgatoren wie das Molkenprotein -Lactoglobulin einen Proteinfilm an der Öl/Wasser-Grenzfläche, dessen Widerstandskraft gegenüber mechanischer Beanspruchung zur Emulsionsstabilität beiträgt. Die Widerstandskraft des Proteinfilms hängt wiederum von den molekularen Wechselwirkungen der angrenzenden Proteine an der Öl/Wasser- Grenzfläche ab, die durch die Proteinstruktur in der wässrigen Phase bestimmt werden.
Aufgrund von Limitationen in der bisher etablierten Analytik von Proteinstrukturen an der Öl/Wasser-Grenzfläche, konnte die Stabilität des Grenzflächenfilms noch nicht systematisch mit der Proteinstruktur während des Grenzflächenstabilisierungsprozesses verknüpft werden.
Um die Proteinfilmstabilität an Öl/Wasser-Grenzflächen zu verstehen und kontrollieren zu können, ist das Ziel dieser Arbeit, den Einfluss der Proteinstruktur auf die (I) Migration durch die Wasserphase, die (II) Adsorption an der Öl/Wasser-Grenzfläche und die (III) Bildung eines Proteinfilms systematisch zu untersuchen. Hierfür wurde die Struktur des Modellproteins -lg in der wässrigen Phase durch Veränderung der Umgebungsbedingungen (pH-Wert, Ionenstärke) und unter hydrostatischer Hochdruckbehandlung gezielt modifiziert. Eine umfassende experimentelle Analytik erlaubte die Charakterisierung der Protein-Nettoladung, der Entfaltung der Sekundärstruktur, der Oberflächenhydrophobizität und des Molekulargewichts. Um strukturelle Veränderungen von -lg nach der Adsorption an der Öl/Wasser-Grenzfläche systematisch zu detektieren, wurde eine in situ FTIR-Methode etabliert. Die Proteinstruktur wurde im Zusammenhang mit dem Grenzflächenverhalten und der Filmstabilität während lateraler Kompression und Expansion, Scherung und Dilatation evaluiert.
Bei hoher Dilatation reduzierte die langsame (I) Migration von elektrostatisch abstoßenden Dimeren die Besetzung von freien Bereichen der Grenzfläche und folglich die Stabilität des Proteinfilms. Die Ausbildung starker intermolekularer Wechselwirkungen während der (III) Proteinfilmbildung, erhöhten die Filmstabilität gegenüber lateraler Kompression und Expansion, Scherung und kleiner Dilatationsdeformation. Eine ausgeprägte Entfaltung an der Öl/Wasser-Grenzfläche und eine hohe Oberflächenhydrophobizität verstärkten die intermolekularen Wechselwirkungen zwischen adsorbierten Proteinen und führten unabhängig von der Nettoladung der Proteine zu der höchsten Proteinfilmstabilität. Eine starke Reduktion der Grenzflächenspannung nach der (II) Adsorption resultierte nicht zwangsläufig in einer hohen Filmstabilität. Dies wurde am Beispiel von elektrostatisch abgeschirmten Proteinen klar, bei denen zwar die höchste Adsorptionsrate, jedoch die geringste Filmstabilität aufgrund geringer Wechselwirkungen beobachtet wurde.
Die Ergebnisse dieser Dissertation erweitern das mechanistische Verständnis der Proteinfilmstabilität an Öl/Wasser-Grenzflächen durch die Kombination von fundamentalen Erkenntnissen über die Proteinstruktur und den Stabilisierungsmechanismen an der Grenzfläche. Diese Arbeit stellt einen vielversprechenden Ansatz zur ganzheitlichen Analyse des Einflusses der Proteinstruktur auf die Stabilität von Proteinfilmen an Grenzflächen vor, sodass maßgeschneiderte emulsionsbasierte Lebensmittel mit hoher kinetischer Stabilität erzeugt werden können.
List of Figures ... IX List of Tables ... XV List of Abbreviations and Symbols ... XVI
1 Motivation and Objective ... 1
2 Theoretical Background ... 6
2.1 Globular Proteins in Water ... 6
2.1.1 Interactions of Globular Proteins ... 6
2.1.2 Structural Characterization of -Lactoglobulin ... 7
2.1.3 Structural Modification of -Lactoglobulin ... 9
2.2 FTIR Analysis of Globular Proteins ... 12
2.3 Model Systems to Describe the Interfacial Stabilization Process and Interfacial Film Stability of Globular Proteins at Oil/Water-Interfaces ... 14
3 Publications ... 21
Manuscript I ... 23
I-1 Abstract ... 24
I-2 Introduction ... 25
I-3 Materials and Methods ... 27
I-4 Results ... 32
I-5 Discussion ... 36
I-6 Conclusion ... 39
Manuscript II ... 41
II-1 Abstract ... 42
II-2 Introduction ... 43
II-3 Materials and Methods ... 45
II-4 Results and Discussion ... 49
II-5 Conclusion ... 58
Manuscript III ... 61
III-1 Abstract ... 62
III-2 Introduction ... 62
III-3 Materials and Methods ... 64
III-4 Results ... 66
IV-2 Introduction ... 77
IV-3 Material and Methods ... 79
IV-4 Results ... 83
IV-5 Discussion ... 88
IV-6 Conclusion ... 94
Manuscript V ... 97
V-1 Abstract ... 98
V-2 Introduction ... 99
V-3 Materials and Methods ... 101
V-4 Results ... 104
V-5 Discussion ... 110
V-6 Conclusion ... 116
4 General Discussion ... 117
4.1 Structural Modification of -Lactoglobulin in the Bulk Water Phase ... 117
4.2 FTIR Analysis of Proteins at the Oil/Water-Interface ... 120
4.3 Stabilization Process of Oil/Water-Interfaces ... 121
4.3.1 Stage (I): Migration Through the Bulk Water Phase ... 122
4.3.2 Stage (II): Adsorption at the Oil/Water-Interface ... 123
4.3.3 Stage (III): Interfacial Protein Film Formation ... 124
4.4 Protein Film Stability at the Oil/Water-Interface ... 126
4.5 Relation Between Protein Structure, Interfacial Stabilization and Protein Film Stability ... 128
5 Concluding Remarks and Outlook ... 131
References ... 135
Annex ... 159
List of Conference Contributions ... 159
List of Additional Publications ... 160
Supplementary Data Manuscript II ... 161
Supplementary Data Manuscript III ... 167
Supplementary Data Manuscript IV ... 168
Supplementary Data Manuscript V ... 170
Figure 1: -lactoglobulin monomer at neutral pH with the secondary structure elements -sheets, -turns, -helix and random coil structures.
... 8 Figure 2: Interfacial stabilization process with (I) migration, (II) adsorption and (III) protein film formation of -lactoglobulin at the oil/water-interface and the subsequent mechanical stress, resulting in low film stability due to low intermolecular interactions. ... 15 Figure 3: Deformation of interfacial protein film with lateral compression and expansion in the Langmuir trough, shear deformation in the shear rheology and dilatational deformation in the pendant drop analysis. ... 18 Figure 4: Interfacial stabilization process with (I) migration, (II) adsorption, (III) film formation and resulting protein film stability against mechanical stress in dependence of the structural modification of -lg in the bulk water phase. Simulated protein pictures are taken from manuscript I & V. ... 130 Figure I-1: Graphical abstract of manuscript I. ... 24 Figure I-2: Drop volume ( ) and evaluation of protein adsorption via linear fl(x) and exponential fit fe(x) of the interfacial tension ( ) of -lactoglobulin at pH 7NaCl against time via two-fluid needle experiments (n = 3, n = 1 illustrated for clarity). (A) 0.000 wt% unoccupied interface with estimation of difference in interfacial tension
u, (B) 0.001 wt% interfacial preoccupation with analysis of difference in interfacial
p1 p2 and (C) 0.010 wt% interfacial preoccupation with the three stages migration through bulk phase (I), adsorption (II) and interfacial rearrangement (III) in accordance with Beverung et al. (1999). ... 28 Figure I-3: Setup of molecular dynamics simulation of -lactoglobulin at pH 7 and pH 9,
approaching to an unoccupied or preoccupied oil/water-interface. A
of -lactoglobulin to the oil/water-interface is varied in each simulation step. B: -lactoglobulin in black circle approaches an oil/water-interface with a preoccupation level of 0.0000 nm-2, 0.0135 nm-2 and 0.0135 nm-2. ... 31 Figure I-4: Stages (I) and (II) of the interfacial stabilization process and estimation of the stabilisation state of -lactoglobulin at pH 7, pH 7NaCl and pH 9 at interfacial preoccupation level of 0.000, 0.001 and 0.010 wt% against MCT-oil via two-fluid needle experiments (n = 3). A: Analysis of lag time ta (I), calculated from the point of addition protein solution until the drop of interfacial tension. B: Evaluation of adsorption rate (II) via slope k of exponential decrease in interfacial tension after addition of protein solution. C: Estimation of interfacial stabilization state via delta
u p before and after adding protein solution. Different letters (a – e) show significant differences between the values (p < 0.05). ... 32 Figure I-5: Interaction forces between -lactoglobulin and MCT-oil interface as function of the distance at pH 7 (A) and pH 9 (B) for different interfacial preoccupation levels (0.0000, 0.0135 and 0.0303 nm-2, n = 3). ... 34 Figure I-6: Force maps, indicating the interaction force at different -lactoglobulin orientation to the MCT-oil interface for different interfacial preoccupation levels (0.0000, 0.0135 and 0.0303 nm-2) and pH values (pH 7 and 9). ... 35
Figure I-7: Graphical illustration of migration through bulk phase (I), adsorption at oil/water-interface (II) and protein film formation (III) of the interfacial stabilization process of -lactoglobulin at pH 7, pH 7NaCl and pH 9. ... 38 Figure II-1: Graphical abstract of manuscript II. ... 42 Figure II-2: (A) FTIR second derivative spectra of Amide I band (n = 3, n = 1 illustrated) and (B) the amount of band intensity at 1631 cm-1 (intramolecular -sheets) and at 1616 cm-1 (intermolecular -sheets) of reference (–) and high hydrostatic pressure-treated (100 – 600 MPa) -lactoglobulin in the bulk water phase (n = 3). (C) MD-simulation of the amount of intramolecular -sheets of high hydrostatic pressure- treated -lactoglobulin at 600 MPa over time and decompression to atmospheric pressure (n = 2, n = 1 illustrated). Different letters (a – e, A – E) represent statistically significant differences (n = 3, p < 0.05). ... 50 Figure II-3: (A) CD spectra (n = 3, n = 1 illustrated) and (B) the calculated number of -helices and unordered structure of reference (–) and high hydrostatic pressure- treated (600 MPa) -lactoglobulin in the bulk water phase (n = 3). (C) MD-simulation of the number of -helices and unordered structures of high hydrostatic pressure- treated -lactoglobulin at 600 MPa over time (n = 2, n = 1 illustrated). Different letters (a, b and A, B) represent statistically significant differences (n = 3, p < 0.05).
... 51 Figure II-4: (A) Extrinsic fluorescence spectra (n = 3, n = 1 illustrated) and (B) calculated hydrophobicity of reference (–) and high hydrostatic pressure-treated (100 – 600 MPa) -lactoglobulin in the bulk water phase (n = 3). (C) MD-simulation of the solvent accessible surface of high hydrostatic pressure-treated -lactoglobulin at 600 MPa over time (n = 2, n = 1 illustrated). Different letters (a – d) represent statistically significant differences (n = 3, p < 0.05). ... 52 Figure II-5: Molecular dynamic simulation of (A) reference -lactoglobulin with a closed -barrel and of (B) high hydrostatic pressure-treated -lactoglobulin at 600 MPa with a partly “open” (unfolded) -barrel, both in the bulk water phase (n = 2, n = 1 illustrated). (C) Migration of water molecules inside the -barrel of high hydrostatic pressure-treated -lactoglobulin at 600 MPa in the bulk water phase. ... 52 Figure II-6: (A) Extinction density against sedimentation coefficient of reference (–) and high hydrostatic pressure-treated -lactoglobulin at 600 MPa in the bulk water phase measured by analytical ultracentrifuge (n = 2, n = 1 illustrated). 2D mass spectrometry of (B) reference (–) and (C) high hydrostatic pressure-treated -lactoglobulin at 600 MPa in the bulk water phase. ... 54 Figure II-7: (A) Lag time, (B) amount of slope of decreasing interfacial tension of the oil/water-interface and (C) difference in interfacial tension of reference (–) and high hydrostatic pressure-treated (100 – 600 MPa) -lactoglobulin measured by pendant drop analysis (n = 3). Different letters (a – c) represent statistically significant differences (n = 3, p < 0.05). ... 56 Figure III-1: (A) Interfacial tension of -lg solutions (0.01 – 1.00wt% in water) in MCT-
oil as function of time (n = 3, n = 1 illustrated) and values of asymptotes of interfacial tension (colored area in A) as function of protein content (B) analyzed by pendant drop and exponentially plotted function (c) with asymptote . ... 67
Figure III-2: (A) Extrinsic fluorescence spectra of protein emulsions with 0.10 – 1.00wt%
-lg content (solid lines, protein content is referred to protein content in water fraction of emulsion) and 1.0wt% -lg in water (dashed line, n = 3, n = 1 illustrated). (B) Emission maxima (colored area in A) as function of protein content with linear fit Imax,E. ... 68 Figure III-3: FTIR subtraction method to remove (A) SDS bands at approx. 1250 cm-1
(colored area) from SDS emulsion spectra SDSE with SDS in water spectrum SDSW to obtain SDS difference spectra SDSE,D and to further remove (B) MCT-oil band at approx. 1750 cm-1 (colored area) from -lg emulsion spectra -lgE to obtain an -lg spectrum -lgE,D with Amide I and II bands at approx. 1500 – 1700 cm-1 (colored area). ... 69 Figure III-4: (A) FTIR spectra -lgE,D of the Amide I and II region for protein emulsions with 0.10 – 1.00wt% -lg content (solid lines) and 1.0wt% -lg in water (dashed line, n = 3, n = 1 illustrated). (B) Peak intensity ratio PIR of Amide I/Amide II (colored area in A) against -lg content (protein content is referred to protein content in water fraction of emulsion) with a double asymptotic function PIR(c) and asymptotes PIR0 and PIR . ... 70 Figure III-5: (A) Second derivative FTIR spectra of -lgE,D in the Amide I region with 0.10 – 1.00wt% -lg content in emulsion (solid lines, protein content is referred to protein content in water fraction of emulsion) and 1.00wt% -lg in water (dashed line, n = 3, n = 1 illustrated). (B) Intensity of 1630 cm-1-band of FTIR second derivative spectra (colored area in A) against protein content and exponential fit I(c) with asymptote I and horizontal dashed line for 1.00wt% -lg in water. ... 72 Figure III-6: Whey protein -lg adsorption at oil/water-interfaces as a function of protein content. Equilibrium between adsorbed and bulk protein tends towards adsorbed protein at stage I and is on side of bulk protein at stage III. The critical interfacial region is in stage II; the oil/water-interface is fully occupied and the interfacial film becomes denser upon protein addition. ... 73 Figure IV-2: Absorbance intensity against wavenumber of FTIR second derivative spectra of 0.1wt% -lactoglobulin in water (black symbols) and in emulsion (protein/water/oil ratio of 0.1/94.9/5.0, white symbols) at pH 7 (A), pH 7NaCl (B) and pH 9 (C). ... 84 Figure IV-3: Langmuir trough analysis of pressure-area isotherms I-A during compression (black symbols) and expansion (white symbols) of the oil/water- interface, occupied with 0.1wt% -lactoglobulin at pH 7 (A), pH 7NaCl (B) and 1.0wt%
-lactoglobulin pH 9 (C). Arrows to the left indicate the compression and arrows to the right the expansion of the oil/water-interface. ... 85 Figure IV-4: Interfacial protein film formation with storage modulus G' (black symbols) and loss modulus G'' (white symbols) against time, analyzed by interfacial shear rheology of 0.1wt% -lactoglobulin at pH 7 (A), pH 7NaCl (B) and pH 9 (C) against MCT-oil. Frequency and amplitude were kept constant at ƒS = 0.1 Hz and S = 0.1%.
... 85 Figure IV-5: Frequency sweeps with storage modulus G' (black symbols) and loss modulus G'' (white symbols) against frequency (ƒS = 0.001 – 1 Hz), analyzed by interfacial shear rheology of 0.1wt% -lactoglobulin at pH 7 (A), pH 7NaCl (B) and pH 9 (C) against MCT-oil. Amplitude was kept constant at S = 0.1%. ... 86
Figure IV-6: Amplitude sweeps with storage modulus G' (black symbols) and loss modulus G'' (white symbols) against amplitude ( S = 0.01 – 100%), analyzed by interfacial shear rheology of 0.1wt% -lactoglobulin at pH 7 (A), pH 7NaCl (B) and pH 9 (C) against MCT-oil. Frequency was kept constant at ƒS = 0.3%. Grey area corresponds to linear viscoelastic region (LVE) calculated from 5% deviation of G'.
... 86 Figure IV-7: Lissajous plots with change in interfacial tension against relative change in drop area / analyzed by interfacial dilatational rheology of 0.1wt% - lactoglobulin at pH 7 (A), pH 7NaCl (B) and pH 9 (C) against MCT-oil at different deformation amplitudes; (1) 0.95%, (2) 5.60% and (3) 11.35%. Frequency was kept constant at ƒD = 0.01%. ... 87 Figure V-1: Graphical abstract of manuscript V. ... 98 Figure V-2: Structural analysis of 0.1wt% reference -lg (grey circles) and hydrostatic pressure-treated -lg at 600 MPa (blue squares) in emulsion (protein/water/oil ratio of 0.1/94.9/5.0); (A) FTIR second derivative spectra in Amide I region (n = 3, n = 1 illustrated) with a decrease in -helices at approx. 1651 cm-1 and intramolecular - sheets at approx. 1628 cm-1 and (B) extrinsic fluorescence analysis at an excitation wavelength of 390 nm with a maximum at approx. 466 nm (n = 3). ... 105 Figure V-3: (A) Pressure-area isotherms -A of reference -lg (grey) in accordance with Kieserling et al. (2021b) and hydrostatic pressure-treated -lg at 600 MPa (blue) measured by Langmuir trough analysis during compression and expansion of the oil/water-interface (n = 2, n = 1 illustrated). (B) Simulated and measured EPR spectra of TB differently partitioned into the three phases. (C) Rotational correlation time C (microviscosity) of individual TB populations and (D) order parameter S (microstructural order) of the protein at the oil/water-interface and the oil phase in emulsions containing reference -lg (grey) and hydrostatic pressure-treated -lg at 600 MPa (blue). Parameters were determined by line-shape analysis of EPR spectra.
Different letters indicate significant differences in the samples (n = 3, p < 0.05). .. 106 Figure V-4: (A) Decrease in interfacial tension of 0.1wt% reference -lg (grey) and hydrostatic pressure-treated -lg at 600 MPa (blue) against MCT-oil, measured by pendant drop system over time (n = 3, n = 1 illustrated). The horizontal dashed line represents the interfacial tension of water against MCT-oil. (B) Protein film formation of 0.1wt% reference -lg (grey circles) and hydrostatic pressure-treated -lg at 600 MPa (blue squares) with interfacial storage modulus G' (dark symbols) and loss modulus G'' (light symbols) against MCT-oil (n = 3) as a function of time, measured with interfacial shear rheology at constant frequency and amplitude (ƒS = 0.1 Hz, 0 = 0.1%). ... 107 Figure V-5: (A) Amplitude sweep ( D = 0.92 – 11.26%) of 0.1wt% reference -lg (grey circles) and hydrostatic pressure-treated -lg at 600 MPa (blue squares) with storage modulus E' (dark symbols) and loss modulus E'' (light symbols) against MCT-oil (n = 3), measured by interfacial dilatational rheology. The frequency was kept constant at ƒD = 0.01%. (B) Lissajous plots with change in interfacial tension against change in drop area A/A0 at the deformation amplitudes (1) 3.68%, (2) 7.43%
and (3) 11.26%. ... 108
Figure V-6: (A) Amplitude sweeps ( S = 0.01 – 100%) of 0.1wt% reference -lg (grey symbols) and hydrostatic pressure-treated -lg at 600 MPa (blue symbols) with interfacial storage modulus G' (dark symbols) and loss modulus G'' (light symbols) against MCT-oil (n = 3), measured by interfacial shear rheology. The frequency was kept constant at ƒS = 0.3%. Maximum amplitude of loss modulus G'' fluctuate around zero and are not displayed in a logarithmic scale. (B) Elastic and (C) viscous Lissajous plots, calculated by shear stress against either strain or strain rate at the amplitudes (1) 4.62%, (2) 21.40% and (3) 99.40%. ... 110 Supplementary data S II-1: High hydrostatic pressure (p) and temperature (T) during the treatment time of 10 min. ... 161 Supplementary data S II-3: Residual analysis of SV-AUC data. Upper part shows measured sedimentation profiles (data points) and numerical solutions of Lamm’s equation (straight lines). Lower part visualizes random residuals, which confirms the validity of the model. The data was created using GUSSI. ... 163 Supplementary data S II-4: Amount of band intensity of FTIR second derivative spectra at 1645 cm-1 (unordered structure) and at 1655 cm-1 ( -helix) of reference (–) and high hydrostatic pressure-treated (100 – 600 MPa) -lactoglobulin in the bulk water phase (n = 3, n = 1 illustrated). Different letters (a – e, A – C) represent statistically significant differences (n = 3, p < 0.05). ... 163 Supplementary data S II-5: Calculated percentage of -sheets and turns from CD spectra of reference (–) and high hydrostatic pressure-treated (600 MPa) -lactoglobulin in the bulk water phase. Different letters (a, b and A, B) represent statistically significant differences (n = 3, p < 0.05). ... 164 Supplementary data S II-7: Molecular dynamic simulation of (A) reference -lg with Trp19 facing the inside of the protein structure and (B) high hydrostatic pressure- treated -lg at 600 MPa with an exposed Trp19 towards the bulk water phase. ... 165 Supplementary data S II-8: Overall interaction potential as a function of the separation distance for -lg monomers and -lg dimers. The interaction potential was calculated based on DLVO interactions. ... 166 Supplementary data S III-1: (A & C) Extrinsic fluorescence spectra of protein emulsions with 0.1 – 1.0wt% -lg content (continuous lines) and 1.0wt% -lg in water (dotted line) treated with ANS with excitation wavelength 390 nm and emission wavelength scan of 420 – 540 nm (n = 3, n = 1 illustrated for clarity). (B) Emission maxima against protein content and emission maxima of 1.0wt% -lg in water max,W. (C) Peak intensity ratio of approx. 470/530 nm peak of extrinsic fluorescence emission spectra as function of protein content in -lg emulsions from 0.1 – 1.0wt% protein and value of -lg in water (dotted line). ... 167 Supplementary data S III-2: (A & B) Non-reproducible FTIR subtraction method to obtain an adsorbed -lg spectrum by subtraction of plain water and MCT-oil spectrum from -lg emulsion spectrum. (C & D) Box plot of oil droplet distribution of analyzed -lg emulsions from 0.1 – 1.0wt% protein and 0.2wt% SDS as well as intensity of Amide I band plotted against median of oil droplet distribution with no correlation.
... 168 Supplementary data S IV-1: Reduced osmotic pressure R against protein content cP, analyzed with membrane-osmometry of -lactoglobulin at pH 7 (A), pH 7NaCl (B) and
Supplementary data S IV-2: FTIR second derivative spectra band wavenumbers and band intensities in Amide I band region of 0.1wt% -lactoglobulin in water and in emulsion (protein/water/oil ratio of 0.1/94.9/5.0) at pH 7, pH 7NaCl and pH 9. ... 169 Supplementary data S IV-3: Extrinsic fluorescence emission intensity against wavelength of 0.1wt% -lactoglobulin in emulsion (protein/water/oil ratio of 0.1/94.9/5.0) at pH 7 (A), pH 7NaCl (B) and pH 9 (C). ... 169 Supplementary data S IV-4: Oil droplet distribution of 0.1wt% -lactoglobulin in emulsion (protein/water/oil ratio of 0.1/94.9/5.0) at pH 7 (A), pH 7NaCl (B) and pH 9 (C). ... 169 Supplementary data S V-1: High hydrostatic pressure (p) and temperature (T) during the treatment time of 10 min. ... 170 Supplementary data S V-2: Interfacial shear rheological frequency sweeps of 0.1wt%
reference -lg (grey circles) and hydrostatic pressure-treated -lg at 600 MPa (blue squares) with interfacial storage modulus G' (dark symbols) and loss modulus G'' (light symbols) in the range of ƒS = 0.001 – 1 Hz. ... 170 Supplementary data S V-3: (A) Relative proportion of TB and (B) hyperfine splitting constant aN (micropolarity) in the different phases of emulsions containing reference -lg (grey columns) and hydrostatic pressure-treated -lg at 600 MPa (blue columns). Both parameters were determined by line-shape analysis of EPR spectra. Different letters indicate significant differences in the samples (n = 3, p < 0.05). ... 170
Table 1: Amino acid composition of whey protein -lactoglobulin (in accordance with Farrell et al., 2004) ... 7 Table 2: Overview of the generated protein structures in the bulk water phase with simulated -lactoglobulin pictures taken from manuscript I & II. ... 118 Table I-1 -potential, particle size (radius r), hydrophobicity R and calculated lag time tc with standard deviation of -lactoglobulin at pH 7, pH 7NaCl and pH 9 for preoccupation levels (0.000, 0.001 and 0.001 wt%). Different letters (a, b, c) in a column show significant differences between the values (n = 3, p < 0.05). ... 33 Table II-1: Molecular weight and second virial coefficient B’, conducted by membrane-osmometry as well as -potential of reference and high hydrostatic pressure-treated -lactoglobulin ( -lg) in the bulk water phase. Different letters (a, b) in a column show significant differences between the values (n = 3, p < 0.05). ... 55 Table V-1: Interfacial shear and dilatational parameters of pressure-treated (600 MPa) and reference -lg. Different letters indicate significant differences in a row (n = 3, p <
0.05). ... 109
Wavenumber
A Drop area
aN Hyperfine splitting parameter
ANS 8-anilinonaphthalene-1-sulfonic-acid AUC Analytical ultra centrifugation cb Bulk protein concentration
CD Circular dichroism
CIC Critical interfacial concentration CMC Critical micellar concentration
d Relative weight
Dc Diffusion coefficient
E' Interfacial dilatation rheological storage (elastic) modulus E'' Interfacial dilatation rheological loss (viscous) modulus E* Interfacial dilatation rheological complex modulus EPR Electron paramagnetic resonance
FTIR Fourier-transform infrared spectroscopy
ƒD Frequency of dilatation
G' Interfacial shear rheological storage (elastic) modulus G'' Interfacial shear rheological loss (viscous) modulus G* Interfacial shear rheological complex modulus
h+1 Low field peak
h0 Middle field peak
IR Infrared
k Highest slope of decreasing interfacial tension
kB Boltzmann constant
LMW Low molecular weight
LVE Linear viscoelastic
Lw Line shape
m Mass
MCT Medium-chain-triacylglycerol
MS Mass spectrometry
NLVE Non-linear viscoelastic
pH 7HP High hydrostatic pressure treatment at pH 7 pH 7NaCl pH value of 7 and addition of 0.1 M NaCl
PIR Peak intensity ratio
r Radius
S Order parameter
SDS Sodium-dodecyl-sulfate
SV Sedimantation velocity Temperature
ta Analytical lag time
tan Interfacial shear rheological loss factor (G''/G') tan Interfacial dilatation rheological loss factor E''/E')
tc Calculated lag time
tt Theoretical lag time
UV/Vis Ultraviolett/visible vm Vibration frequency
VMD Visual molecular dynamics
W Broadening constant
Interfacial concentration
Dynamic viscosity
Wavelength
µ Reduced mass
Frequency
c Rotational correlation time Mean drop area
-lg -lactoglobulin
D Amplitudes of dilatation Interfacial tension
Mean of interfacial tension
Many common food products such as beverages, dairy products, dressings and soups are dispersed systems, in particular emulsions. Nanoemulsions and more complex systems such as multiple-emulsions are increasingly being used as targeted delivery systems for functional food ingredients such as vitamins, flavors and colors in foods (Donsì et al., 2011;
Muschiolik, 2007; Velikov & Pelan, 2008). In the case of oil/water-emulsions, non-polar oil is dispersed as droplets in the polar bulk water phase. During the emulsification process, the interfacial tension between the oil and water is reduced through emulsifiers that adsorb at the oil/water-interface (Bos & van Vliet, 2001; Dickinson, 2010; Foegeding & Davis, 2011). As reviewed by Lam and Nickerson (2013), the food industry prefers natural high molecular weight emulsifiers like whey proteins over synthetic low molecular weight emulsifiers such as monoglycerides to stabilize the dispersed oil droplets in the bulk water phase. Since the size distribution of the oil droplets determines the characteristic properties of emulsion-based products, e.g., texture and color, a small oil droplet size is aimed for (Dalgleish, 2006). In order to produce the desired oil droplet size distribution, emulsification requires different processes ranging from the more gentle membrane emulsification to a high energy input by using high pressure homogenization (Floury et al., 2000; Spyropoulosa et al., 2011).
The stabilization of oil/water-interfaces with proteins like the aforementioned whey proteins is a complex process. In the neutral bulk water phase, the major whey protein -lactoglobulin ( -lg) has a negative net charge and exhibits a globular structure. Its secondary structure mainly comprises intramolecular -sheets, -turns, random coil structures and a short segment of -helices in the folded state (Creamer et al., 1983). The tertiary structure refers to the conformation of the folded secondary structure elements and corresponds with its surface hydrophobicity, since hydrophobic amino acid residues face towards the inside of the protein structure (Southall et al., 2002; Tanford, 1997). Finally, the quaternary structure describes the association of the monomeric subunit, resulting in a sensitive equilibrium of -lg monomers, dimers and octamers (Sawyer et al., 1999; Taulier
& Chalikian, 2001).
Subsequent interfacial stabilization can be described in simplified terms as a three-staged process (Baldursdottir et al., 2010; Beverung et al., 1999). The protein (I) migrates through the bulk water phase, (II) adsorbs at the oil/water-interface and (III) forms an interfacial protein film. More specifically, the migration of proteins from the bulk to the oil/water- interface in stage (I) is a convection- and diffusion-controlled step that mainly depends on the hydrodynamic radius in the bulk water phase, which is closely linked to the unfolding state and the quaternary structure of the protein (Baldursdottir et al., 2010; Ravera et al., 2005). The migration (I) and adsorption in stage (II) are affected by the increasing amount of protein at the oil/water-interface as a consequence of changes in the concentration gradient (Beverung et al., 1999; Mezzenga & Fischer, 2013; Wahlgren et al., 1997). The adsorption rate depends on the affinity between the protein and the non-polar oil phase and
it is increased by hydrophobic interactions (Baldursdottir et al., 2010; Dickinson, 1998;
Erni, Windhab, & Fischer, 2011; Singh, 2011). By contrast, high electrostatic repulsion and a pronounced steric hindrance between the proteins slows down the adsorption (Dombrowski et al., 2016; Wahlgren et al., 1997). In stage (III), the formation and properties of the interfacial protein film are determined by the interfacial molecular density of the proteins and the unfolding that favors the intermolecular interactions between adsorbed proteins, resulting in a film with viscoelastic properties (Bos & van Vliet, 2001;
Lucassen-Reynders, 1993; Petkov et al., 2000; Sagis & Scholten, 2014). Put simply, the unfolding of the secondary structure, the surface hydrophobicity (tertiary structure), the molecular weight (quaternary structure) and charge determine the interfacial stabilization process of proteins. Due to the high system dynamics, the stabilization process is much more complex during emulsification but the basic steps, as outlined above, still hold true and determine emulsion characteristics and stability.
Besides stabilization, destabilizing phenomena may occur in emulsions, thereby limiting the shelf-life. These result from mechanical stress of the oil/water-interface during subsequent processing as well as from the thermodynamical instability. At the micro-level, the mechanical stress on an oil/water-interface can occur in the form of lateral compression and expansion as well as shear and dilatational deformation (Benjamins et al., 2006; Krägel
& Derkatch, 2010; Murray, 1997; Murray et al., 2002; Petkov et al., 2000). Both, lateral compression and expansion and shear stress appear as two-dimensional deformation along the oil/water-interface, while the composition of the interfacial area is not affected. By contrast, dilatational deformation appears as a three-dimensional compression and expansion, and therefore it includes changes in the composition of the interfacial area, i.e., through de- and adsorption of protein molecules. The resistance of an interfacial protein film against mechanical stress is closely linked to its viscoelastic properties that comprise an elastic part accounting for the solid character and a viscous part accounting for the liquid character (Benjamins et al., 2006; Bos & van Vliet, 2001). A high stability of the interfacial protein film requires a high elastic-to-viscous ratio (Dickinson, 1998; Freer et al., 2004;
Lam & Nickerson, 2013; Murray, 1998; Rühs et al., 2012) and can be achieved through many and strong attractive intermolecular interactions between adjacent proteins. In particular, covalent disulfide bonds as well as non-covalent electrostatic interactions, hydrogen bonds, hydrophobic associations and van der Waals interactions increase the elasticity, and thus the protein film stability against mechanical stress (Murray, 1998; Rühs et al., 2012; Ziegler & Foegeding, 1990). At the macro-level, there are several forms of emulsion destabilization mechanism, all of which result in phase separation into oil and water, since this is the most thermodynamically stable state (Capek, 2004). Dispersed oil droplets tend to flocculate, merge together (coalesce) and cream (Bos & van Vliet, 2001;
Dalgleish, 1997b; Dickinson, 1998; Sriniv et al., 1999; Tcholakova, Denkov, Sidzhakova, Ivanov, & Campbell, 2005). In this context, the repulsion between two dispersed oil droplets is highly desirable, resulting from the electrostatic repulsion between equal
intermolecular interactions between the adsorbed adjacent proteins at the oil/water- interface should be predominantly attractive to build strong protein films that resist the aforementioned mechanical stress. By contrast, the intermolecular interactions between two approaching oil droplets and therefore between interfacial proteins films should be predominantly repulsive (Capek, 2004). It becomes apparent that a conflict of interests in respect to the molecular interactions exists.
The molecular interactions also define the protein structure. Since interactions are not static and vary with the ambient conditions, the protein structure is easily affected throughout the interfacial stabilization process. Moreover, the structural properties of proteins at the oil/water-interface depend on the protein structure in the bulk water phase (Dalgleish, 1996; Y. Fang & Dalgleish, 1997; Husband et al., 2001; Tcholakova, Denkov, Sidzhakova, et al., 2006). Despite the fact that each stage of the interfacial stabilization process has already been analyzed individually, none of these studies have connected all three stages, while systematically analyzing the molecular interacions and protein structure to explain the resulting film stability. Consequently, several authors have emphasized the need for comprehensive and systematic investigations that range from the protein in the bulk water phase to its film stability against mechanical stress (Krägel & Derkatch, 2010; Murray, 2011; Wilde, 2000). Furthermore, recent studies emphasize that not only the molecular interactions determine the structure of the adsorbed proteins, but also vice versa, namely that the protein structures exercise a strong influence on the intermolecular interactions, and therefore the viscoelastic properties of the protein film (Dickinson, 2011; Keppler et al., 2018; Lam & Nickerson, 2013; Murray, 2011; Rühs et al., 2012; Sagis & Scholten, 2014; Zhai et al., 2010). In consequence, the understanding of the complex interplay of molecular interaction and the structural properties of the proteins at the oil/water-interface is a necessary prerequisite to understand the mechanisms behind the protein film stability against mechanical stress.
This thesis aims to investigate the influence of the protein structure on the interfacial stabilization process, the (I) migration, (II) adsorption and (III) protein film formation and the resulting protein film stability at the oil/water-interface. Consequently, the systematic investigation of the interfacial behavior requires the controlled modification of the protein structure. Structural modifications can be achieved by varying the surrounding bulk water conditions, that is pH value, ionic strength and high hydrostatic pressure (Considine et al., 2007; Liu et al., 2009; Majhi et al., 2006; Mercadante et al., 2012; Sakurai & Goto, 2002;
Sawyer & Kontopidis, 2000; Taulier & Chalikian, 2001). The variation in the pH value determines the protonation state of the amino acid residues within the primary structure of the protein and it further determines the protein net charge, the unfolding of the structure and the surface hydrophobicity due to an exposition of hydrophobic amino acid residues (Lindman et al., 2006; McKenzie & Sawyer, 1967; Mercadante et al., 2012; Sakurai &
Goto, 2002). Monovalent salts like NaCl shield electrostatic interactions by attaching to oppositely charged amino acid residues (Aymard et al., 1996; Renard et al., 1998). High hydrostatic pressure treatment varies the unfolding of proteins by compression of the protein structure, resulting in changes on all structural levels (Considine et al., 2007). Given
various controversial discussions have ensued concerning the structure of -lg (Considine et al., 2007; Liu et al., 2009; Majhi et al., 2006; Mercadante et al., 2012; Sakurai & Goto, 2002; Sawyer & Kontopidis, 2000; Taulier & Chalikian, 2001). Another challenge for the systematic interpretation of the protein structure, interfacial stabilization process and resulting protein film stability is the lack of precise methods for analyzing the structural changes upon adsorption at the oil/water-interface (Dalgleish, 2006). As highlighted by Zhai et al. (2013), in most analytical methods this is due to the superimposition of the measurement signals of the protein and its surrounding oil and water phases, often caused by light scattering effects of the dispersed oil droplets. At present, the surface hydrophobicity of the proteins at the oil/water-interface can be estimated with fluorescence analysis and the net charge of the adsorbed proteins by determining the -potential (Donsmark & Rischel, 2007; Sakuno et al., 2008; Tcholakova et al., 2005; Zhai et al., 2011). Fourier-transform infrared spectroscopy (FTIR) generally is a highly sensitive method to detect changes in the secondary structure but there is no method available to obtain accurate protein signals at the oil/water-interface of emulsions so far.
To fulfill the aim of the present thesis, the structure of model protein -lg in the bulk water phase was intentionally modified by pH, ionic strength and high hydrostatic pressure to induce changes in the folding of secondary structure, hydrophobicity, molecular weight and charge (manuscript I & II). In this context, the analysis of structural changes during adsorption at the oil/water-interface required the optimization of an FTIR method (manuscript III). While the first and second stages of the interfacial stabilization process were characterized in manuscript I and II, the discussion of the third stage and the film stability are part of manuscript IV and V. Regarding the interfacial stabilization process and protein film stability, the following hypotheses were defined.
Since the migration in stage (I) of the interfacial stabilization process is controlled by both diffusion and intermolecular interactions, the combination of a high hydrodynamic diameter, caused by a partly unfolded structure and a dimeric conformation, with an electrostatic repulsion of charged proteins reduces the migration through the bulk water phase. By comparison, an increased charge is compensated by a low hydrodynamic radius and a dimeric conformation by electrostatic shielding, resulting in no effect on the migration (manuscript I & II).
In stage (II) of the interfacial stabilization process, the adsorption rate is increased by both an increased surface hydrophobicity, which enhances the affinity to the hydrophobic oil phase, as well as electrostatic shielding of the proteins due to the reduced intermolecular repulsion, whereas the steric difference between monomeric and dimeric conformations does not influence the adsorption rate (manuscript I & II).
In stage (III) of the interfacial stabilization process, electrostatic shielding favors a high interfacial molecule density but also impedes further unfolding of adsorbed proteins, resulting in weak interactions and low elastic properties of the interfacial protein film. An increase of the surface charge will lower the interfacial molecular density due to electrostatic repulsion of the adsorbed proteins, while a high hydrophobicity favors the formation of intermolecular interactions and thus elasticity (manuscript IV & V).
The protein film stability against lateral compression and expansion, shear and small dilatational deformation of the oil/water-interface correlates with pronounced elastic properties and thus the attractive intermolecular interactions of the adsorbed proteins during (III) film formation. By contrast, at high dilatational deformation, the protein film stability is sustained due to a fast (I) migration of the proteins through the bulk water phase rather than a high (II) adsorption rate due to the role of diffusional exchange with the unoccupied oil/water-interface during oscillation (manuscript IV & V).
This thesis aimed to investigate the interfacial behavior of the model protein -lg in dependence of its structure. Therefore, the first subchapter gives an overview of the interactions that contribute to the globular structure of -lg in water as well as tools in order to modify the protein structure. The next subchapter focuses on the principles of FTIR, since one of the goals of this work was to establish an FTIR method sensitive enough to characterize minor structural changes of proteins at the oil/water-interface. In the third subchapter the interfacial stabilization process as well as the analysis of the film stability against mechanical stress is put forth.
2.1 Globular Proteins in Water
The following subchapters describe the main intra- and intermolecular interactions that occur between the amino acid residues of globular proteins such as -lg. Moreover, it describes the protein structure in the bulk water phase and how the protein structure can be modified by bulk water or processing conditions.
2.1.1 Interactions of Globular Proteins
In order to reach minimal free energy, the structure of globular proteins as -lg adapt to the surrounding conditions through favorable molecular interactions in the bulk water phase.
Intramolecular interactions refer to interactions between amino acid residues within a protein molecule, while intermolecular interactions designate interactions between two molecules (i.e., protein-protein, protein-water). Molecular interactions in proteins include covalent bonds, steric effects, electrostatic interactions, hydrogen bonds, van der Waals and hydrophobic interactions (Israelachvili, 2011). Due to their significant role in this thesis, the interactions are briefly described in the following subchapter based on the explanations of Israelachvili (2011).
Covalent bonds are short-range bonds (binding length of approx. 0.2 nm) with a binding strength of up to 950 kJ mol-1. They involve two atoms with similar electronegativities that share electron pairs in dependence of their electronic configuration. Electrostatic (Coulomb) interactions are non-covalent by nature and they occur between charged or polar groups, i.e., between permanent or induced dipoles. They strongly vary in their binding length and show high binding strengths of up to 800 kJ/mol. Most of the other interactions that occur, such as steric interactions, hydrogen bonds and van der Waals interactions, originate from electrostatic interactions. Steric interactions are repulsive by nature and they result from the local electrostatic repulsion of electrons in the outer shells of atoms, which hence result in steric hindrance between approaching molecules (Walstra, 2001). Hydrogen bonds are non-covalent electrostatic interactions between a lone pair of electrons of an
positively polarized and exhibit a small radius. However, the strength of the hydrogen bonds is strongly affected by the electronegativity of the hydrogen bond donor, the orientation of the donor and acceptor groups, the binding length and the surrounding phase.
Typically, hydrogen bonds have bond strengths between 10 and 40 kJ mol and short binding lengths of approx. 0.2 nm. Van der Waals interactions include non-covalent interactions between two dipoles, induced dipoles or dispersion forces, and therefore they occur between all kinds of atoms. Van der Waals interactions are weak bonds with binding strengths between 2 and 15 kJ mol , whereas their long-range character enables binding lengths up to 10 nm. Hydrophobic interactions are entropically-driven associations of non- polar groups in a hydrophilic surrounding phase like water that is referred to as hydrophobic effect (Meyer et al., 2006; Southall et al., 2002; Tanford, 1997). When non-polar molecules are incorporated in water, the water molecules rearrange in ordered cage-like structures around the non-polar molecules, to obtain as many hydrogen bonds as possible. Since the arrangement of the water molecules causes an energetically unfavorable decrease in entropy, the non-polar molecules tend to associate and interact via van der Waals interactions.
2.1.2 Structural Characterization of -Lactoglobulin
The structure of dissolved globular proteins in water is divided into the four levels of primary, secondary, tertiary and quaternary structure. The primary structure refers to the covalently linked amino acids within the peptide backbone of the protein. Thus, the type, number and sequence of amino acids define the secondary, tertiary and quaternary structural levels through specific inter- and intramolecular interactions (summarized in Chapter 2.1.1) -lg, the protein is composed of 162 amino acids (Sawyer &
Kontopidis, 2000), whose residues show aromatic, hydrophobic, hydrophilic uncharged, positively- and negatively-charged properties, as summarized in Table 1.
Table 1: Amino acid composition of whey protein -lactoglobulin (in accordance with Farrell et al., 2004).
Amino acid -lg Amino acid -lg
Aromatic
Phenylalanine Phe 4
Positively charged
Arginine Arg 3
Tyrosine Tyr 4 Histidine His 2
Tryptophan Tpr 2 Lysine Lys 15
Hydrophilic uncharged
Serine Ser 7 Negatively
charged
Aspartic acid Asp 10
Threonine Thr 8 Glutamic acid Glu 16
Asparagine Asn 5
Hydrophobic
Alanine Ala 15
Glutamine Gln 9 Valine Val 9
Cysteine Cys 5 Isoleucine Ile 10
Glycine Gly 4 Leucine Leu 22
Proline Pro 8 Methionine Met 4
The secondary structure refers to highly ordered -helices and -sheets, stabilized by intramolecular hydrogen bonds between carbonyl (C=O) and amino (N-H) groups of the peptide backbone, as well as unordered random coil structures. The secondary structure of -lg mainly comprises nine intramolecular -sheets (approx. 50%, referred to strand A-I), eight of them (A-H) organized into a -barrel, -turns (approx. 10%), a short -helix segment (approx. 10%) and additional parts with random coil structures with approx. 30%
(Casal et al., 1988; Dong et al., 1996; Papiz et al., 1986; Tolkach & Kulozik, 2007). The strands of the -sheets are connected by loops that are referred to as -turns when consisting of four to five amino acid residues.
The tertiary structure refers to the conformation of the folded secondary structure elements.
It is stabilized by intramolecular interactions of the amino acid residues, including hydrogen bonds, hydrophobic, electrostatic and van der Waals interactions, as well as covalent disulfide bonds (Lumry & Eyring, 1954; Southall et al., 2002; Tanford, 1997). In particular -lg has five thiol groups, four of which are involved in disulfide bonds (Cys66-Cys160 and Cys106-Cys119) and one (Cys121) incorporated in the interior of the protein, the latter capable of forming intermolecular disulfide bonds (Sakurai & Goto, 2002; Alexander Tolkach & Kulozik, 2007). The tertiary structure corresponds with the surface hydrophobicity, since hydrophobic and aromatic amino acid residues face towards the inside of the protein structure (specifically inside secondary structure elements such as -sheets and -helices) the due to the hydrophobic effect (Southall et al., 2002; Tanford, 1997).
Finally, the quaternary structure of the globular protein describes the specific arrangement of secondary and tertiary structure, and hence the association of monomeric subunits. A -lg exhibits a molecular weight of 18.4 kDa (Renard et al., 1998) and is depicted in Figure 1.
Figure 1: Molecular dynamics simulation1 of a -lactoglobulin monomer at neutral pH with the secondary structure elements -sheets, -turns, -helix and random coil structures.
