Document [original]

Interfacial properties of saponins from

Quillaja saponaria Molina and their

functionality in dispersed systems

vorgelegt von

Sandra Böttcher, M.Sc.

geb. in Karl-Marx-Stadt

von der Fakultät III – Prozesswissenschaften

der Technischen Universität Berlin

zur Erlangung des akademischen Grades

Doktor der Ingenieurwissenschaften

- Dr.-Ing –

genehmigte Dissertation

Promotionsausschuss:

Vorsitzender: Prof. Dr. Lothar W. Kroh

Gutachter: Prof. Dr. Stephan Drusch

Gutachterin: Prof. Dr. Cornelia Rauh

Gutachter: Kamil Wojciechowski, PhD DSc

Tag der wissenschaftlichen Aussprache: 28. April 2017

Berlin, 2017

Substitution of Acknowledgements

Sandra Böttcher Technische Universität Berlin

Acknowledgments

So this is it: I finished my thesis! You may not anticipate how much work this was, but

that’s ok. It was all worth it and I would certainly do it again! This section will give you a

small hint how many people were involved in it and to whom I am deeply thankful to.

Let’s start with the department Food Technology and Food Material Science at Tech-

nische Universität Berlin, Germany where I performed most of my research. I want to thank

the Friedrich-Naumann-Foundation for Freedom and the Bundesministerium für Bildung

und Forschung for the financial support of this thesis. Besides the financial support, I am

very grateful for my time as a scholar because I met amazing people, went abroad for con-

ferences and research stays and had the opportunity to actively participate and educate

myself and let others benefit from my knowledge. Being a scholar in the FNF is more than

money, once you are a scholar you become part of the Naumann family. I made so many

great friends, participated in awesome events, broadened my perspective and learned so

many new things.

I want to especially thank my supervisor Prof. Dr. Stephan Drusch for his guidance and

his support throughout the time of my thesis. I know I have been a challenging PhD student

but it’s fair to say the same about you as a supervisor and I am very thankful to you that

you pushed me to my personal limits. I grew with every heated discussion and tried every

day to become a better researcher. Thanks for giving me the chance to make my PhD. It is

not easy for graduates from a University of Applied Sciences to get the chance for starting

a PhD. Also I want to thank Prof. Dr. Cornelia Rauh and Prof. Dr. Kamil Wojciechowski

for agreeing to examine my thesis.

I want to thank Ingredion, especially Ines Fuhlrott for supplying the Quillaja saponin

and for fruitful discussions and cooperation. And thanks to Scott Osborne and Procter &

Gamble for offering a position in research as a scientist. This prospective helped me

through the last weeks and I am looking forward to start a new chapter after the end of my

thesis.

During my PhD, I had the honor to collaborate with other scientific research groups and

want to thank Prof. Dr. Matteo Scampicchio and his group for a great research stay in Bol-

zano trying to get reasonable ITC measurement. Although it did not work out, we had a

great time! I also like to thank Dr. Julia K. Keppler for the generous supply of the β-LG

and her scientific help on the fluorescence experiments. I also want to thank Prof. Dr.

Acknowledgements

Sandra Böttcher Technische Universität Berlin

Sascha Rohn and Dr. Valeria Reim for teaching me the extraction of saponins and HPTLC

at their department at Universität Hamburg.

My PhD would have been less fun and enjoyable without the people of the department

Food Technology and Food Material Science. Thanks to all of my co-workers and espe-

cially to Kenneth. What’s better than working with friends? Sharing an office with them.

You were the best office buddy and I will miss our laughs. Laughing about all the cruel

stuff the lab did to us, made it far easier to cope with it. Anja, why didn’t you join us earlier?

You will be such an amazing professor one day! Rocio, my crazy Spanish girl. Thank you

for bringing so much sunshine to Berlin. With you I always had humongous laughs. Helena,

we had so little time together and I wished it would have been more. You are such a great

person with a caring and passionate soul and you will rock your PhD. Janine, Addi and

Freddi thank you for establishing so many methods I was able to use and your welcoming

arms when I arrived.

It was a pleasure having Ariane Muth, Hanna von Heymann and Irina Reifschneider as

my students but the greatest was reserved for the end: Marina Eichhorn. You were abso-

lutely amazing and I really loved working with you! Keep on going, there is so much you

can achieve.

Sport ist Mord und Knieverletzungen sind ätzend. Danke an alle meine Taekwondoins,

die mich immer wieder aufgebaut haben! Der schwarze Gürtel wird kommen, auch wenn

es noch etwas dauern wird.

Während meiner Doktorarbeit hatte ich die besten Freunde, die man sich wünschen

kann. Ihr wart immer für mich da, besonders in den letzten Wochen vor der Abgabe haben

mich eure Nachrichten, Treffen und Anrufe wiederaufgebaut und mir geholfen fertig zu

werden. Julian, sei il migliore. Ohne dich wäre das Phdlife nicht halb so großartig gewesen.

Danke für tausende lustige Bilder, Chats, Ablenkungen, wenn ich eigentlich hätte schreiben

müssen, die unzähligen Besuche in Berlin und Treffen anderswo in der Welt. Frie, mein

partner in crime, wenn es mal wieder ums „recherchieren“ ging. Mo, ich möchte dir sagen

(3 Tage später) du bist großartig und ich freue mich wirklich, dass wir so nah beieinander

wohnen. Marlene, ich bin immer noch so dankbar, dass du mich als Drill Instructor mara-

thonfit gemacht hast und die letzten Jahre so viel Sonnenschein in mein Leben gebracht

hast. Kenneth und Christoph, wenn‘s mit der Diss doch nicht klappt, könnt ihr als Comedy

Duo auf jeden Fall den ganz großen Durchbruch schaffen! Sandra, mit dir habe ich so viele

tolle Locations kennengelernt und Events erlebt. Wenn‘s mir mal reicht, komme ich vorbei

zum Schaukeln.

Substitution of Acknowledgements

Sandra Böttcher Technische Universität Berlin

Die wichtigsten Menschen aber nun zum Schluss: meine Familie. Mama, Papa, Omi, Opi,

Conny und Lutz. Ich liebe euch und bin euch unendlich dankbar für eure konstante und

bedingungslose Unterstützung in jeder Situation. Danke Mama, für deine unglaublich klu-

gen Weisheiten. Danke Papa, dass du mich zu einer Kämpferin gemacht hast. Hinter jedem

starken Kind stehen unglaubliche Eltern, ich kann euch nicht genug danken, dass ihr mich

zu der erzogen habt, die ich nun bin. Danke Omi und Opi, ihr habt mich seit meiner Kind-

heit jederzeit unterstützt und wer hat schon so coole Großeltern wie euch?!

Steffen – du bist der Wichtigste von allen. Ohne dich und deine unendliche Liebe wäre

meine Welt einfach nur grau und langweilig. Wir sind diesen Weg von Anfang an gemein-

sam gegangen. Jetzt beginnt ein neues Kapitel, möge es eins von Hunderten weiteren sein.

Abstract

Sandra Böttcher Technische Universität Berlin

Abstract

The aim of the present thesis was to connect interfacial properties of saponins at aqueous

interfaces with their behavior in dispersed systems. Thereby the influence of changes in pH

and ionic strength and interactions with β-lactoglobulin were discussed using spectroscopic

equipment and knowledge on molecular structure.

Saponins are phytochemicals that can be found in numerous plant species in low con-

centrations. Due to their amphiphilic structure, saponins are surface active and may be used

in dispersed systems like emulsions and foams. Molecular structure of saponins is highly

variable as different aglyone types exist to which a varying quantity and type of sugar res-

idues may be linked. Although it is known that saponins with a triterpenoid aglycone form

viscoelastic films, knowledge on corresponding foam properties is scarce.

In the present thesis six saponins, which differed in molecular structure, were analyzed

with respect to interfacial and foam properties. It was shown that high complex dilational

and shear moduli were a requirement for stable foams but not a guarantee for high stability.

However, it was also shown that the type of aglycone and length of the sugar chains had

the highest impact on foam stability.

A saponin extract from Quillaja saponaria Molina (QS) is already approved for appli-

cation in food products in the EU and US. But until now only little is known about

interactions between QS and food-relevant ingredients like proteins. There has been evi-

dences that QS and β-lactoglobulin (β-LG) form complexes, which affected interfacial

properties. In the present thesis fluorescence experiments showed complex formation in the

bulk and interfacial rheology indicated strong interactions at the air/water-interface. The

interactions between QS and β-LG tremendously increased foam stability. In contrast mix-

tures of QS and β-LG negatively affected emulsion stability by inducing aggregation of oil

droplets. Food systems may not only contain proteins but may also be a mixture of multiple

dispersed phases. It was shown that oil droplet size of the emulsion and pH are key factors

in controlling stability of foamed emulsions. Stability may further be enhanced by sequen-

tially adding QS to a β-LG-emulsion.

Summarizing the results, experimental data showed that molecular structure of saponins

distinctly affects foam stability. In addition, it was shown that mixing QS with β-LG may

lead to synergistic or antagonistic effects in dispersed systems. However, it is necessary to

perform experiments, which further deepen our understanding on the relationship of mo-

lecular structure and interfacial properties as well as behavior in dispersed systems.

Furthermore, future research should focus on the underlying intermolecular interactions

between proteins and QS to explain interfacial properties of mixtures of both.

Zusammenfassung

Sandra Böttcher Technische Universität Berlin

Zusammenfassung

Ziel dieser Dissertation war es grundlegende Zusammenhänge zwischen Grenzflächen-ei-

genschaften von Saponinen und deren Verhalten in dispersen Systemen zu verstehen.

Zusätzlich wurde auf Grundlage des molekularen Aufbaus der Saponine und mithilfe von

spektroskopischen Verfahren der Einfluss von pH und Ionenstärke sowie die Interaktionen

mit β-Laktoglobulin (β-LG) auf die Eigenschaften von dispersen Systemen diskutiert.

Saponine sind sekundäre Pflanzenstoffe, die in geringen Konzentrationen in einer Viel-

zahl von botanischen Quellen vorkommen. Durch ihre amphiphile Struktur sind Saponine

grenzflächenaktiv und können demnach in dispersen Systemen, wie Schäumen und Emul-

sionen zur Stabilisierung eingesetzt werden. Es gibt zahlreiche Saponinderivate, die sich in

der Art des Aglykongerüsts sowie der Anzahl und Länge der verknüpften Zuckerketten

unterscheiden. Der Zusammenhang zwischen molekularer Struktur und Grenzflächenei-

genschaften sowie Schaumeigenschaften ist bisher nur wenig erforscht.

Es wurden die Grenzflächen- und Schaumeigenschaften von sechs Saponinen aus unter-

schiedlichen botanischen Quellen untersucht, die sich in der Art des Aglykons und der

Länge sowie Anzahl der Zuckerreste unterscheiden. Es wurde gezeigt, dass die Ausbildung

eines stark viskoelastischen Films eine notwendige Bedingung aber keine Garantie für eine

hohe Schaumstabilität ist. Die Art des Aglykons und die Länge der Zuckerketten waren

dabei die strukturellen Merkmale, die den größten Einfluss auf die Schaumstabilität hatten.

Im Gegensatz dazu hatte die Anzahl der verknüpften Zuckerketten (Mono- oder Bidesmo-

sidisch) nur einen geringen Einfluss.

Bisher ist nur ein saponinreicher Extrakt aus Quillaja saponaria Molina (QS) für den

Einsatz in Lebensmitteln zugelassen. Die Wechselwirkungen von QS mit Proteinen sind

bisher nur wenig erforscht. Es gab in der Vergangenheit Hinweise auf eine Komplexbil-

dung zwischen QS und β-LG. Fluoreszenzexperimente bestätigten eine Komplexbildung

im Bulk und grenzflächenrheologische Untersuchungen zeigten intermolekulare Wechsel-

wirkung an der Luft/Wasser-Grenzfläche. Die Wechsel-wirkungen zwischen QS und β-LG

führten zu einer deutlich erhöhten Schaumstabilität. Im Gegensatz dazu führten Mischun-

gen aus QS und β-LG in Emulsionen zur Aggregation von Öltröpfchen, welche die

Stabilität der Emulsionen deutlich erniedrigte. Einige Lebensmittel sind Mehrphasengemi-

sche, wie z.B. aufgeschäumte Emulsionen. Es wurden zum ersten Mal Schlüsselfaktoren

zur Kontrolle der Stabilität von aufgeschäumten QS-Emulsionen definiert. Dazu zählen die

Öltröpfchengröße der Emulsion und der pH-Wert. Die Stabilität kann außerdem durch die

sequentielle Zusage von QS zu einer β-LG Emulsion erhöht werden.

Zusammenfassung

Sandra Böttcher Technische Universität Berlin

Es wurde in dieser Dissertation gezeigt, dass die molekulare Struktur von Saponinen

einen signifikanten Einfluss auf die Schaumstabilität hat und dass Mischungen mit β-Lak-

toglobulin zu synergistischen und antagonistischen Effekten in dispersen Systemen führen

können. Es wird mehr Forschung benötigt um den Zusammenhang zwischen molekularer

Struktur von Saponinen und deren Grenzflächeneigenschaften sowie Eigenschaften in dis-

persen Systemen zu verstehen. Des Weiteren werden Untersuchungen benötigt um die

Interaktionen mit Proteinen auf molekularer Ebene zu erklären.

Table of contents

Sandra Böttcher Technische Universität Berlin

Table of contents

List of tables ................................................................................................................... XIV

List of figures ................................................................................................................... XV

List of abbrevations ........................................................................................................ XIX

Publications ........................................................................................................................ 23

Conference contributions ................................................................................................... 25

1. Introduction ................................................................................................................. 27

2. Literature review ......................................................................................................... 33

2.1. Composition and characterization of saponin extracts ...................................... 33

2.2. Self-assembly and micellar structure ................................................................ 36

2.3. Linking molecular structure and behavior at aqueous interfaces ...................... 37

2.3.1. Interfacial configuration of saponins at the air/water-interface .................... 38

2.3.2. Supramolecular interfacial configuration of Quillaja saponins at the

air/water-interface ......................................................................................... 41

2.3.3. Impact of the hydrophobic phase on interfacial properties of various

saponins ........................................................................................................ 43

2.4. Interactions of saponins with other (food) components .................................... 44

Manuscript I ..................................................................................................................... 47

I-1 Abstract ............................................................................................................. 48

I-2 Introduction ....................................................................................................... 49

I-3 Materials and Methods ...................................................................................... 51

I-3.1 Fourier transform infrared spectroscopy (FTIR) .......................................... 52

I-3.2 Conductivity measurements.......................................................................... 53

I-3.3 Interfacial tension measurements using Wilhelmy plate .............................. 53

I-3.4 Short-term adsorption of saponin extracts on the air-water interface ........... 54

I-3.5 Foaming and foam stability .......................................................................... 56

I-3.6 Determination of foam structure with analysis of brightness profiles and

foam pictures ................................................................................................ 57

I-4 Results and discussion ....................................................................................... 57

I-4.1 CMC determination and fitting of interfacial tension isotherms using the

modified Frumkin model .............................................................................. 57

I-4.2 Short-term adsorption using two-fluid needle experiments .......................... 58

I-4.3 Foaming, foam stability and foam structure ................................................. 61

I-4.4 Influence of pH and salt on interfacial tension and foam properties ............ 63

I-5 Conclusions ....................................................................................................... 68

Table of contents

Sandra Böttcher Technische Universität Berlin

Manuscript II ................................................................................................................... 69

II-1 Abstract ............................................................................................................. 70

II-2 Introduction ....................................................................................................... 71

II-3 Material and methods ........................................................................................ 73

II-3.1 Purification of saponin extract ...................................................................... 73

II-3.2 Fluorescence quenching of β-LG by the presence of QS ............................. 74

II-3.3 Dynamic interfacial tension measurements, dilational rheology, short-term

adsorption and sequential two-fluid needle experiments.............................. 75

II-3.4 Shear rheology .............................................................................................. 76

II-3.5 Foaming, foam stability and foam structure ................................................. 76

II-4 Results ............................................................................................................... 77

II-4.1 Interactions in the bulk determined by fluorescence quenching .................. 77

II-4.2 Interactions of QS and β-LG at the interface ................................................ 78

II-4.3 Short- and midterm adsorption of mixtures of QS and β-LG ....................... 79

II-4.4 Dilational rheology of mixtures of β-LG/QS ............................................... 80

II-4.5 Shear rheology .............................................................................................. 81

II-4.6 Foam properties ............................................................................................ 82

II-5 Discussion ......................................................................................................... 83

II-6 Conclusion ......................................................................................................... 89

Manuscript III .................................................................................................................. 91

III-1 Abstract ............................................................................................................. 92

III-2 Introduction ....................................................................................................... 93

III-3 Material and methods ........................................................................................ 96

III-3.1 Materials ....................................................................................................... 96

III-3.2 Drop shape analysis of adsorption, dynamic interfacial tension and dilational

rheology ........................................................................................................ 96

III-3.3 Interfacial shear rheology at the oil/water-interface ..................................... 98

III-3.4 Emulsification and emulsion stability .......................................................... 98

III-3.5 Determination of oil droplet size distribution and ζ-potential ...................... 98

III-4 Results ............................................................................................................... 99

III-4.1 Adsorption and dynamic interfacial tension at the oil/water-interface as

determined by drop shape analysis ............................................................... 99

III-4.2 Dilational rheology determined by droplet oscillation and analysis of non-

linear phenomena of interfacial layers ........................................................ 101

III-4.3 Shear rheology of interfacial layers ............................................................ 105

III-4.4 Oil droplet size distribution of emulsions as determined by static light

scattering ..................................................................................................... 106

III-4.5 ζ-potential of the emulsion droplets............................................................ 107

III-4.6 Visual analysis of emulsion stability after a storage time of 7 days ........... 108

III-5 Discussion ....................................................................................................... 109

III-6 Conclusions ..................................................................................................... 117

Table of contents

Sandra Böttcher Technische Universität Berlin

Manuscript IV ................................................................................................................ 119

IV-1 Abstract ........................................................................................................... 120

IV-2 Introduction ..................................................................................................... 121

IV-3 Material and methods ...................................................................................... 125

IV-3.1 Preparation of emulsions ............................................................................ 125

IV-3.2 Oil droplet size distribution, ζ-potential and interfacial tension of the

emulsions .................................................................................................... 126

IV-3.3 Preparation and characterization of foamed emulsions .............................. 127

IV-4 Results and discussion ..................................................................................... 128

IV-4.1 Impact of homogenization parameters on oil droplet size distribution of

emulsions .................................................................................................... 128

IV-4.2 Emulsion properties and stability of foamed QS-emulsions affected by the

oil droplet size ............................................................................................. 130

IV-4.3 Influence of pH on ζ-potential and stability of foamed QS-emulsions ...... 135

IV-4.4 Emulsification of a binary mix of QS and β-lactoglobulin and the influence

on the stability of foamed emulsions .......................................................... 138

IV-5 Conclusion ....................................................................................................... 144

6. General discussion ..................................................................................................... 145

6.1. The impact of structural features on interfacial properties of saponins from

different botanical sources ............................................................................... 146

6.2. Synergistic and antagonistic effects of QS/β-LG-mixtures on stability of

dispersed systems ............................................................................................ 149

6.2.1. Synergistic effect of QS/β-LG-mixtures on foam properties ..................... 150

6.2.2. Antagonistic effect of QS/β-LG-mixtures on emulsion stability ................ 153

6.2.3. Maximizing stability of foamed emulsions ................................................ 156

7. Concluding remarks and outlook ............................................................................ 161

References ........................................................................................................................ 165

Annex 179

A-I Materials and devices ............................................................................................ 179

A-II Characterizing foaming, foam stability and foam structure ................................. 181

A-III Validation of foam analysis ................................................................................ 184

Curriculum Vitae ............................................................................................................ 187

XIV

List of tables

Sandra Böttcher Technische Universität Berlin

List of tables

Table I-1 Origin, purity and general chemical properties of the six saponin extracts .................. 51

Table I-2 Fitting parameters of the experimental data using the Frumkin model ......................... 58

Table I-3 Short- and midterm adsorption parameters of six saponin extracts at twofold CMC

pH 5 .............................................................................................................................. 59

Table I-4 Results from foam experiments of QS, GYP, TS, ESC and GA at twofold CMC,

pH 5 .............................................................................................................................. 62

Table I-5 CMCcond, CMCSFT, slope below (kbelow), slope above (kabove) the CMC and ratio of

both (kbelow/kabove) for QS, GYP, ESC and GA; for TS and TT no breaking point was

detected ........................................................................................................................ 64

Table II-1 Dynamic interfacial tension σ of β-LG-solutions and β-LG/QS-mixtures measured

for 20 min with pendant drop analysis ......................................................................... 79

Table III-1 Dynamic interfacial tension (IFT) of β-LG-solutions and β-LG/QS-mixtures

measured for 20 min with pendant drop analysis at the oil/water-interface ............ 100

Table IV-1 d50 of 0.3 % QS-emulsions (5 % MCT-oil) in relation to number of passes and

homogenization pressure; bold numbers refer to chosen parameters for experiment

on foaming of emulsions ............................................................................................ 126

Table A-1 Origin, purity and general chemical properties of the used saponin extracts ............. 179

Table A-2 Devices ....................................................................................................................... 180

Table A-3 Parameters to characterize foaming, foam stability and foam structure;

n - time point during the analysis; / - none ................................................................ 183

Table A-4 Variation of parameters to characterize foaming, foam stability and foam

structure depending on QS concentration; n - time point during the analysis ........... 185

List of figures

Sandra Böttcher Technische Universität Berlin

List of figures

Figure I-1 Surface pressure Π versus square root of the drop age t1/2 at the air/water interface

of QS (◆), GYP (▲), TS (×), ESC (●), GA (■) and TT (+) from different

botanical origins at pH 5, twofold CMC ...................................................................... 60

Figure I-2 Foam characterization with the parameters a) foaming speed (kf) and b) foam

density at f-max (fDen,f-max) and (c) foaming pictures after 80 and 1800 s of foaming

of QS, GYP, TS, ESC and GA at twofold CMC .......................................................... 61

Figure I-3 FTIR spectra of C═O binding region (1718-1731 cm-1) of QS (◆), GYP (■),

TS (▲), ESC (●), GA (+) and TT (x) ......................................................................... 64

Figure I-4 Influence of pH and ionic strength on interfacial tension σ at 0.5-fold CMC .............. 65

Figure I-5 Influence of decreased pH and increased ionic strength by the addition of NaCl

on foaming speed (kf), foam decay after 3600s (f3600s) and foam structure (BDm,600s,

BDw,600s and fden-fmax) of A) QS, B) GYP, C) TS and D) ESC at 0.5-fold CMC;

pH 5 - black solid line (reference values set as 1), pH 3 – gray solid line,

100 mM – gray dotted line, 500 mM – gray dashed line .............................................. 66

Figure I-6 Foam pictures of GA at 0.5-fold CMC after 80s of start of foaming at pH 5

(upper left) and pH 3 (upper right) and TS at 0.5-fold after 600 s of start of

foaming at 0 mM (lower left) and 500 mM (lower right) ............................................ 67

Figure II-1 Fluorescence measurements on the interactions of β-LG and QS, A) absorption spectra

between 250-300 nm of pure β-LG (solid line), pure QS (dashed line)

and mixture of β-LG+QS, which was corrected for QS adsorption (dotted line),

B) Cogan-Plot of quenching of β-LG by QS with P=β-LG-concentration,

α=fraction of free binding sites and Lt=QS-concentration ......................................... 77

Figure II-2 Interfacial tension σ in relation to the drop age t for the injection of 0.15 % QS

into a droplet of 10 mM phosphate buffer (◆) and sequential adsorption of 1 %

β-LG at the air water interface with injection indicated by the arrow of

0.15 % QS (●) and injection of 10 mM phosphate buffer (○) ................................... 78

Figure II-3 Surface pressure Π versus square root of the drop age t1/2 at the air/water interface

of 0.005 % QS (+), 0.005 % β-LG (◇), 0.01 % β-LG (△), 0.05 % β-LG (○),

0.1 % β-LG (□); filled symbols indicate β-LG is mixed with 0.005 % QS ............... 79

Figure II-4 Interfacial dilational modulus E* in relation A) the drop age t and B) to the

surface pressure Π of 0.005 % QS (+), 0.005 % β-LG (◇), 0.01 % β-LG (△),

0.05 % β-LG (○), 0.1 % β-LG (□); filled symbols indicate β-LG is mixed with

0.005 % QS ................................................................................................................ 80

Figure II-5 Complex shear modulus G* in relation the interface age t for of 0.005 % QS (+),

0.005 % β-LG (◇), 0.01 % β-LG (△), 0.05 % β-LG (○), 0.1 % β-LG (□);

filled symbols indicate β-LG is mixed with 0.005 % QS .......................................... 81

Figure II-6 Foam results of QS (red), β-LG (blue) and mixtures of QS and β-LG (violet) for

A) remaining foam height after 3600 s and B) foam density at fmax in relation to

β-LG concentration .................................................................................................... 82

XVI List of figures

Sandra Böttcher Technische Universität Berlin

Figure III-1 Results of A) lag-time and B) surface pressure after 2 s of adsorption (Π2s) in

relation to the β-LG concentration of 0.005 % QS (◇), β-LG (○) and mixtures

of 0.005 % QS and β-LG (⚫) at the MCT-oil/water-interface ............................... 99

Figure III-2 Interfacial dilational modulus E* in relation to β-LG-concentration determined

by dilational oscillation at f=0.1 Hz, 2.8 % amplitude of 0.005 % QS (◇), β-LG (○)

and mixtures of 0.005 % QS and β-LG (●) at the MCT-oil/water interface. Error

bars represent 10 % deviation, which was determined in previous experiments. . 101

Figure III-3 Phase angle Φ in relation to area change ΔA/A0 determined by dilational

oscillation at f=0.1 Hz at the MCT-oil/water interface of 0.005 % QS (+),

0.005 % β-LG (◇), 0.01 % β-LG (△), 0.05 % β-LG (○), 0.1 % β-LG (□);

filled symbols indicate β-LG is mixed with 0.005 % QS ..................................... 102

Figure III-4 Interfacial tension σ versus area change ΔA/A0 (Lissajous-plots) determined by

dilational oscillation at f=0.1 Hz at different amplitudes 1.4, 2.8, 4.2 and 7.0 %

at the MCT-oil/water interface of a) 0.005 % QS (+), b) 0.01 % β-LG (△),

c) 0.005 % QS+0.005 % β-LG (◆), d) 0.005 % QS+0.05 % β-LG (△),

e) 0.005 % QS+0.05 % β-LG (●), f) 0.005 % QS+0.1 % β-LG (□) ..................... 103

Figure III-5 S-factor versus amplitude during expansion (left panel) and compression

(right panel) of a droplet at the oil/water-interface determined by dilational

oscillation at f=0.1 Hz of 0.005 % QS (+),0.005 % β-LG (◇), 0.01 % β-LG (△),

0.05 % β-LG (○), 0.1 % β-LG (□); filled symbols indicate β-LG is mixed

with 0.005 % QS ................................................................................................... 104

Figure III-6 Complex (G*), elastic (G’) and viscous (G’’) shear moduli for 0.005 % QS;

0.005, 0.01, 0.05 & 0.1 % β-LG and 0.005 % QS mixed with 0.005, 0.01,

0.05 & 0.1 % β-LG (from left to right) at the MCT-oil/water-interface

measured with f=1 Hz, deformation=0.1% after 9 h of film formation ................ 105

Figure III-7 Oil droplet size of emulsions prepared with 0.005 % QS (1st boxplot),

0.005…0.1 % β-LG (2nd to 5th boxplot) and mixtures of 0.005 % QS and

0.005…0.1 % β-LG (6th to 9th boxplot) with upper whisker representing d90,

upper box end d75, dash in the box d50, lower box end d25 and lower whisker d10 106

Figure III-8 ζ-potential in relation to the β-LG concentration of emulsions (600 bar,

4 passes, 5 % MCT-oil) with 0.005 % QS (◇), β-LG (○) and mixtures of

0.005 % QS and β-LG (●) ..................................................................................... 107

Figure III-9 Photographic images of emulsions prepared with 0.005 % QS; 0.005, 0.01,

0.05 & 0.1 % β-LG and 0.005 % QS mixed with 0.005, 0.01, 0.05 & 0.1 %

β-LG (from left to right) after 7 days of storage ................................................... 108

Figure III-10 Fluorescence measurements on the interactions of β-LG and QS showing

results of the Cogan-Plot with P=β-LG-concentration, α=fraction of free

binding sites and Lt=QS-concentration. Adapted from Böttcher et al. (2015)

with permission from Elsevier. ............................................................................. 110

Figure III-11 Microscopic images of emulsions after 7 days of storage prepared with

0.005 % QS and A) 0.005 % β-LG, B) 0.01 % β-LG, C) 0.05 % β-LG and

D) 0.1 % β-LG the black bar on the lower right represents 25 µm ....................... 115

List of figures

XVII

Sandra Böttcher Technische Universität Berlin

Figure IV-1 Schematic illustration of the relationship between the stability of foamed

emulsions and their oil droplet size. Please refer to the text for appropriate

explanation of the different ranges. ....................................................................... 123

Figure IV-2 Influence of (A) number of on the oil droplet size distribution of 0.3 %

QS-emulsion (200 bar, 5 % MCT-oil) with upper whisker representing d90,

upper box end d75, dash in the box d50, lower box end d25 and lower whisker d10

and (B) influence of homogenization pressure on the oil droplet size

distribution of 0.3 % QS-emulsion (2 passes, 5 % MCT-oil)................................ 128

Figure IV-3 Double logarithmic plot of the relationship between d50 and homogenization

pressure of a 0.3% QS-emulsion (5 % MCT-oil) for 1 pass (■), 2 (▲), 3 (●)

and 4 passes (♦) ..................................................................................................... 129

Figure IV-4 Emulsion properties with respect to (A) interfacial tension σ (□) and foaming

speed v (◇) and (B) properties of the foamed emulsions described by the

median of the brightness distribution BDm after 1 h measurement (△) in

relation to the median (d50) of 0.3 % QS-emulsions with range 1, 2 and 3.

Lines are only guide to the eye. Please refer to the text for appropriate

explanation of the different ranges. ....................................................................... 132

Figure IV-5 Brightness profiles of foamed emulsions of 0.3 % QS with d50 of A) 0.2 µm,

600 bar, 3 passes; B) 0.3 µm, 500 bar, 2 passes; C) 0.4 µm, 300 bar, 2 passes;

D) 0.5 µm, 100 bar, 3 passes; E) 0.6 µm, 200 bar, 2 passes; F) 0.8 µm, 100 bar,

4 passes; G) 1 µm, 100 bar, 3 passes; H) 1.2 µm, 100 bar, 2 passes; I) 2.1 µm,

50 bar, 3 passes ...................................................................................................... 133

Figure IV-6 Specific interfacial a rea Aspec In relation to d50 of 0.3% QS-emulsions .................. 134

Figure IV-7 Foam profile showing total height in relation to measuring time for foamed

emulsions (5 % MCT-oil, d50=0.5 µm) prepared at A) pH 3 B) pH 5 and

C) pH 7. A was analyzed for 1800 s, B and C for 3600 s ..................................... 135

Figure IV-8 ζ-potential in relation to pH of 0.3 % QS-emulsions (5 % MCT-oil, d50=0.5 µm) . 136

Figure IV-9 Foamed emulsion of 0.3 % QS (5 % MCT-Oil, d50=0.5 µm) after 1800 s with

macroscopic aggregation of oil droplets................................................................ 137

Figure IV-10 ζ-potential (A) and interfacial tension after 150 s (B) of emulsions (0.3 % QS,

5 % MCT-oil, d50=0.5 µm) in relation to β-LG-concentration for QS (○),

β-LG (◇), Mixpre (△) and Mixpost (□) ................................................................... 139

Figure IV-11 Schematic illustration of location of QS (green) and β-LG (violet) in emulsions

(A+C) and foamed emulsions (B+D) whereas Mixpre is illustrated in A+B and

Mixpost in C+D ....................................................................................................... 140

Figure IV-12 Foaming speed of emulsions (0.3 % QS, 5 % MCT-oil, d50=0.5 µm) in relation

to β-LG-concentration for QS (○), β-LG (◇), Mixpre (△) and Mixpost (□) ............ 141

Figure IV-13 Foam profiles showing total height in relation to measuring time for a foamed

emulsion (d50=0.5 µm) prepared with 5 % MCT-oil and (A) 0.3 % QS;

(B) 0.1 % β-LG; (C) 0.2 % β-LG;(D) 0.3 % β-LG; (E)

0.3 % QS+0.1% β-LG (Mixpre); (F) 0.3 % QS+0.2 % β-LG (Mixpre);

(G) 0.3 % QS+0.3% β-LG (Mixpre); (H) 0.3 % QS+0.1% β-LG (Mixpost);

(I) 0.3 % QS+0.2 % β-LG (Mixpost); (J) 0.3 % QS+0.3% β-LG (Mixpost) ............. 142

XVIII List of figures

Sandra Böttcher Technische Universität Berlin

Figure A-1 Foam profile of a foam made from a (A) 0.003 % and (B) 0.01 % QS solution ...... 181

Figure A-2 Derived parameters from foam profile 1 - Time to maximum foaming level –

tfmax [s], 2 - Maximum foaming level – fmax[mm], 3 - Slope of foaming –

kf [mm/s], 4 – Relative remaining foam height– fn%[%],

5 - Foam half-life time – tf1/2[s], 6 - Stability of maximum foam height –

tfmax-5% [s], 7 – relative drainage – dn%[%], 8 - Foam density – fden,n[%],

9 - Foam density at f-max – fden,fmax% [%],

10 - Analysis of image brightness distribution – median BDm,n and width BDw,n ... 182

Figure A-3 Example for histogram of brightness distribution at a specific time point

(rectangle of 1 px width) of a foam made from 0.01 % QS solution after 1800 s ... 184

List of abbrevations

XIX

Sandra Böttcher Technische Universität Berlin

List of abbrevations

%wt Weight percent

A/W Air/water-interface

ATR Attenuated total reflection

BSC Saponin-rich extract from Sapindus mukurossi

CCD Charge-coupled device

CMC Critical micelle concentration

CMCcond Critical micelle concentration determined with conductivity

CnDMPO Alkyldimethylphosphine oxide with alkyl chain (non-ionic surfactant)

Cryo-TEM Cryo-transmission electron microscopy

CTAB Cetyltrimethylammoniumbromid (cationic surfactant)

DATEM Diacteyltartaric esters of monoglycerides

ESC Saponin-rich extract from Aesculus hippocastanum

ESI Electron spray ionization

FAB Fast atom bombardment (ionization method)

FTIR Fourier transform infrared spectroscopy

GA Glycyrrhizic Acid Ammonium Salt from Glycyrrhiza glabra

GRAS Generally regarded as safe

GS Saponin-rich extract from Panax ginseng

GYP Saponin-rich extract from Gypsophia species

HPLC High performance liquid chromatography

HPTLC High performance thin layer chromatography

IFT Interfacial tension

ITC Isothermal titration calorimetry

LC-MS Liquid chromatography coupled with mass spectrometry

MALDI Matrix-assisted laser desorption/ionization

MCT Medium-chain triglyceride

NMR Nuclear magnetic resonance spectroscopy

O/W Oil/water-interface

O/W/A Oil/water/air-interface

QS Saponin-rich extract from Quillaja saponaria Molina

SDS Sodium dodecyl sulfate (anionic surfactant)

SLS Sodium laureth sulfate (anionic surfactant)

TOF Time of flight detector

Triton X-100 Polyethylene oxide with aromatic hydrocarbon or hydrophobic group

(non-ionic surfactant)

Trp Trypthophan

TS Saponin-rich extract from Camellia oleifera Abel

XX List of abbrevations

Sandra Böttcher Technische Universität Berlin

TT Saponin-rich extract from Tribulus terrestris

Tween 80 Polysorbate 80 (non-ionic surfactant)

USDA U.S. Department of Agriculture

β-LG Beta-lactoglobulin

a Interaction parameter

A Area per molecule

Aem Absorption value at the emission wavelength

Aexc Absorption value at the excitation wavelength

b Adsorption equilibrium constant

BDm,n Median of brightness distribution of foam profiles at time point n

BDw,n Width of brightness distribution of foam profiles at time point n

c Bulk concentration

c0 Concentration of surfactant in the bulk solution

d Diameter

Dcalc Calculated diffusion coefficient

Dexp Experimental diffusion coefficient

dx Diameter at x-percentile

E* Interfacial/Complex dilational modulus

E’ Elastic (storage) dilational modulus

E’’ Viscous (loss) dilational modulus

F0 Fluorescence intensity of the initial β-LG-concentration

Fcorr Recalculated fluorescence value

fDen,f-max Percentage of incorporated liquid in the foam

fDen,n Foam density and foam density at f-max

Fmax Fluorescence intensity upon saturation

fn Remaining foam height at time point n

Fobs Measured fluorescence value

G* Interfacial/Complex shear modulus

G’ Elastic (storage) shear modulus

G’’ Viscous (loss) shear modulus

K Adsorption parameter

K’a Apparent affinity constant

List of abbrevations

XXI

Sandra Böttcher Technische Universität Berlin

K’d Apparent dissociation constant

kB Boltzmann constant

kf Foaming speed (start of foaming until maximum foam height (f-max))

Lt QS concentration in fluorescence experiments

n Maximum number of binding sites

n Specific time point in foam analysis

P β-LG concentration in fluorescence experiments

R General gas constant

r Hydrodynamic radius of a spherical molecule

S Strain stiffening ratio

Scom Strain stiffening during compression

Sext Strain stiffening ratio during expansion

T Absolute temperature

t Time

tan δ Dissipation factor (G’’/G’)

β Interaction parameter

ΔA/A Area change

ε Compressibility of the interfacial monolayer

ζ Zeta-potential

η Dynamic viscosity

θ Surface coverage

Π Surface pressure

Π2s Surface pressure 2 s after interfacial tension drop

σ Interfacial tension

σ0 Interfacial tension of water

Φ Phase angle

ω Molar surface area

ω0 Molar area at Π=0

Г Surface concentration of surfactant

Г∞ Maximum surface concentration of surfactant at c0→∞

Publications

Sandra Böttcher Technische Universität Berlin

Publications

Manuscript I

Böttcher, S. and Drusch, S., 2016, “Interfacial Properties of Saponin Extracts and Their

Impact on Foam Characteristics”, Food Biophysics, V. 11, No. 1, pp. 91–100.

The final publication is available at Springer via http://dx.doi.org/10.1007/s11483-

015-9420-5.

Manuscript II

Böttcher, S.; Scampicchio, M.; and Drusch, S., 2016, “Mixtures of saponins and beta-

lactoglobulin differ from classical protein/surfactant-systems at the air-water inter-

face”, Colloids and Surfaces A: Physicochemical and Engineering Aspects,

V. 506, pp. 765–773. doi: http://dx.doi.org/10.1016/j.colsurfa.2016.07.057.

Manuscript III

Böttcher, S.; Keppler, J.; and Drusch, S., 2017, “Mixtures of Quillaja saponin and

beta-lactoglobulin at the oil/water-interface: adsorption, interfacial rheology and

emulsion properties,” Colloids and Surfaces A: Physicochemical and Engineering As-

pects, V. 518, pp. 46–56. doi: http://dx.doi.org/10.1016/j.colsurfa.2016.12.041.

Manuscript IV

Böttcher, S.*; Eichhorn, M.*; and Drusch, S., 2017, “Factors affecting foamed emul-

sions prepared with an extract from Quillaja saponaria Molina: oil droplet size, pH

and presence of beta-lactoglobulin”, Food Biophysics, V. 12, No. 2, pp. 250–260.

doi: http://dx.doi.org/10.1007/s11483-017-9481-8.

Manuscript V

Böttcher, S. and Drusch, S., 2016, “Saponins – self-assembly and behavior at aqueous

interfaces”, Advances in Colloids and Interface Science, V. 243, pp. 105–113.

The final publication is available at Springer via

http://dx.doi.org/10.1016/j.cis.2017.02.008.

*Co-first authorship

Conference contributions

Sandra Böttcher Technische Universität Berlin

Conference contributions

• Böttcher, S. and S. Drusch, presentation at National ISEKI-Workshop on “hot

topics” in the Field of Food Science and Technology: Substitution von nieder-

molekularen Emulgatoren in dispersen Lebensmittelsystemen durch Saponine,

01.07.2014 in Berlin, Germany

• Böttcher, S. and S. Drusch, presentation at 2nd colloquium of the Institute of

Food Science and Technology TU Berlin: Einsatz von Saponinen zur Stabilisier-

ung von dispersen Systemen, 20.10.2014 in Berlin, Germany

• Böttcher, S. and S. Drusch, poster at 3rd colloquium of the Institute of Food Sci-

ence and Technology TU Berlin: Saponins as stabilizing agents in foams,

20.04.2015, Berlin, Germany

• Böttcher, S. and S. Drusch, presentation at SOMATAI-conference:„Quillaja

saponin: An emulsifier unlike common low-molecular weight surfactants,

03.06.16 in Crete, Greece

• Böttcher, S. and S. Drusch, poster at 30th Conference of the European Col-

loid and Interface Society: Quillaja saponin – a promising natural surfactant,

05.09.16 in Rome, Italy

Introduction

Sandra Böttcher Technische Universität Berlin

1. Introduction1

Saponins are natural phytochemicals, which occur in an estimated three out of four plants

species (Hänsel and Sticher 2010). The broad occurrence of saponins in the plant kingdom

can be attributed to their bitter taste, which protects the plant from being eaten by animals.

For a long time, saponins in plant materials were considered undesirable ingredients in

human and animal nutrition because of their bitter taste and undesired side-effects on the

gastro-intestinal system when consumed at high concentrations (Fenwick et al. 1991). As

a phytochemical, saponin content in plants is usually around 1 % or lower (Fenwick and

Oakenfull 1983) but the bark of the Chilean soap bark tree (Quillaja saponaria Molina) has

an exceptionally high saponin content of up to 5 % (Kuznesof and Soares 2005). The com-

position of saponin extracts and their saponin concentration does not only depend on the

plant species but also on the part of the plant, seasonal changes and extraction parameters

(Fenwick et al. 1991; Cheok et al. 2014). When saponins are extracted from plant material,

the resulting extracts are always a mixture of different saponin derivates. For instance ex-

tracts from Quillaja saponaria Molina may contain more than 100 different saponins (Kite

et al. 2004) as well as other anionic residual plant substances (Maier et al. 2015a; Tippel et

al. 2017).

The growing interest on saponins from Quillaja saponaria Molina as adjuvants (Sun et

al. 2009) led to an increasing amount of studies investigating membranolytic and haemo-

lytic properties (Oda et al. 2000; Sparg et al. 2004; Reim and Rohn 2015). For this purpose

interactions of saponins with membrane lipids like DPPC and cholesterol (Demana et al.

2004; Wojciechowski et al. 2014c; Wojciechowski et al. 2016a) and lecithin bilayers

(Wojciechowski et al. 2016b) were studied. Saponins may interact with the membrane cho-

lesterol, which may have adverse effects on cells. The interactions are assumed to originate

from hydrogen bonds formed between sugar residues of saponins and the polar head groups

of the DPPC and cholesterol (Wojciechowski et al. 2016a).

Mixed systems play an important role in foods, but also in many other fields like cos-

metics and especially dispersed systems are of high interest in research and industry. In

dispersed food systems two or more immiscible phases are mixed, which may be of gase-

ous, liquid and/or solid state. When air is dispersed in a continuous liquid phase or liquid

is dispersed in a continuous liquid phase these systems are be referred to as ‘foam’ and

1 Parts of the introduction were published as Manuscript V

Introduction

Sandra Böttcher Technische Universität Berlin

‘emulsion’, respectively. Foams and emulsions (excluding microemulsions) are thermody-

namically unstable systems by definition but meta-stable states may be obtained. To form

and stabilize dispersed systems, surface active constituents are essential. A molecule is re-

ferred to as surface active when hydrophilic and hydrophobic parts are present in the

molecular structure. Low-molecular weight surfactants, various proteins and certain

charged polysaccharides are examples of surface active molecules. Surface active mole-

cules adsorb at interfaces and align their hydrophobic (apolar) and hydrophilic (polar)

structural features according to the affinity of the dispersed and the continuous phase. In an

emulsion, apolar parts of the molecule are moved from the aqueous liquid phase (polar)

and into the apolar oil phase because this is thermodynamically favored. As a result of the

adsorption at the interface, interfacial tension is decreased and free energy of the system is

reduced as described by the Gibbs adsorption equation. Surface active molecules may slow

down coalescence and aggregation of oil droplets in emulsions by electrostatic and steric

repulsion. In foams, adsorbed surface active molecules form an interfacial layer that decel-

erates bubble coalescence, which may be caused by the rupture of the thin liquid film (liquid

film between two adjacent bubbles). In addition, these thin films could be stabilized by the

formation of a viscoelastic network, steric and electrostatic repulsion as well as the Gibbs-

Marangoni mechanism. A detailed description of stabilizing mechanisms of surface active

constituents in foams and emulsions would be beyond the scope of this introduction. The

reader is referred to Sadoc and Rivier (1999), Schramm (2005) and Kralova and Sjöblom

(2009) for more extensive information on stabilizing mechanisms in dispersed systems in

general.

In recent years, researchers regained interest in using saponins in dispersed systems due

to their interfacial activity and unique behavior. Saponins have shown interesting interfacial

properties like formation of a viscoelastic network at aqueous interfaces (Stanimirova et al.

2011; Golemanov et al. 2012; Wojciechowski 2013), formation of micelles (Mitra and

Dungan 1997; Mitra and Dungan 2000) as well as formation of stable foams (Jian et al.

2011; Chen et al. 2010) and emulsions (Yang et al. 2013; Yang and McClements 2013).

Especially the formation of a viscoelastic network is very unusual for such small molecules.

Saponins usually have a molecular weight between 800 to 2,500 Da (Dinda et al. 2010;

Thalhamer and Himmelsbach 2014) and are larger than low molecular weight surfactants,

Introduction

Sandra Böttcher Technische Universität Berlin

which have a molecular weight below 500 Da. Nevertheless, the molecular weight of sap-

onins is lower compared to common proteins like β-lactoglobulin (18,400 Da), β-casein

(24,000 Da) and lysozyme (14,300 Da).

Only few interfacial studies focused on the interfacial properties of saponins from bo-

tanical sources other than Quillaja saponins (extracted from Quillaja saponaria Molina,

QS). Molecular structures of different saponins from various botanical origin were exten-

sively reported and reviewed (Güçlü-Üstündağ and Mazza 2007; Vincken et al. 2007;

Dinda et al. 2010). The structural diversity that may be found in one extract makes it chal-

lenging to work with saponins. Initial studies on the relationship between structural features

(type of aglycone and amount of sugar residues) and interfacial properties e.g. shear and

dilational properties were conducted (Golemanov et al. 2014; Pagureva et al. 2016). Sapo-

nins with a triterpenoid aglycone form high viscoelastic interfacial layers, which exhibit

higher shear and dilational moduli in comparison to saponins with a steroidal aglycone. In

contrast, differences in interfacial properties were not as easily connected to the amount of

linked sugar residues. Although the formation of the viscoelastic network is mainly influ-

enced by the type of aglycone Golemanov et al. (2012) concluded from detailed

experiments on interfacial rheology of QS layers that intermolecular hydrogen bonds be-

tween adjacent sugar residues are responsible for the network formation. It may be

summarized that initial studies revealed some structural features, which are associated with

high shear and dilational moduli but little is known for saponins regarding the relationship

between interfacial rheology and properties in dispersed systems like foams.

Kezwon and Wojciechowski (2014) recently reviewed present state of knowledge on

interactions between Quillaja saponins with food proteins like β-lactoglobulin (β-LG), β-

casein and lysozyme. It is well-known that β-LG forms a viscoelastic network at the air/wa-

ter-interface (Petkov et al. 2000). But it is still controversially discussed whether β-LG

unfolds upon adsorption at the air/water-interface as recently reviewed by Wierenga and

Gruppen (2010). For a long time it was generally accepted that proteins adsorb at the air/wa-

ter-interface and slowly unfold at the interface over time as it was first described by Graham

and Phillips (1979). The unfolding was concluded from dynamic interfacial data because it

takes several hours for β-LG-solutions to reach equilibrium. A rather new explanation is

the ‘colloid approach’ described by Talbot et al. (2000) using the ‘Random Sequential Ad-

sorption model’, which proposes that no or only little unfolding of the β-LG molecules

occurs at the interface. It is assumed that the full adsorption takes very long time, because

Introduction

Sandra Böttcher Technische Universität Berlin

the likeliness for a protein to find an unoccupied space at the interface decreases over time

and slows adsorption. Since the protein molecules at the interface are immobile, a protein

molecule that wants to adsorb at an already occupied space is rejected and diffuses into the

bulk again.

However, it was shown that complexes between Quillaja saponins and proteins influence

interfacial properties and may impact foam properties (Kezwon and Wojciechowski 2014).

But the impact of interactions between QS and β-LG on foam stability as well as interfacial

shear and dilational rheology remains unknown. It may be speculated that a joint interfacial

network between QS and β-LG might be formed, which leads to high viscoelastic moduli

and corresponding high foam stability. The high foam stability due to the formation of a

viscoelastic network discriminates the QS/β-LG-systems and other surfactant/protein-sys-

tems. Surfactants usually stabilize foams by the Gibbs-Marangoni mechanisms and proteins

by a viscoelastic interfacial network. These two possible mechanisms are opposing each

other and usually lead to reduced foam stability in mixed surfactant/protein-systems (Pra-

dines et al. 2009; Lech et al. 2014).

Both QS and β-LG have differing properties at the oil/water-interface, which might in-

fluence interactions between both constituents leading to deviating interfacial and emulsion

properties. In contrast to the air/water-interface the viscoelastic dilational and shear moduli

of QS are distinctively lower at the oil/water-interface (Wojciechowski 2013; Golemanov

et al. 2014). Therefore, interactions with β-LG might not lead to a strong viscoelastic film,

which can withstand deformation and thoroughly stabilize oil droplets against coalescence.

Researchers agree on the considerable unfolding of β-LG at the oil/water-interface and nu-

merous studies characterized adsorption with sophisticated methods (Maldonado-

Valderrama and Patino 2010; Zhai et al. 2013; Zare et al. 2016).

Fundamental studies on interfacial rheology and two-phase dispersed systems like emul-

sions and foams are important to understand mechanisms in more complex systems. The

application of new surfactants in real food products can be challenging, because multiple

variables influence properties of these systems and matrix effects may occur between sur-

factant and food ingredients. Until now there have been various studies on the application

of QS in food systems (Yang and McClements 2013; Ozturk et al. 2014; Zhang et al. 2015;

Zhang et al. 2016). In certain products like mousse and vegan milk foam high stability is

desired. These products contain a mixture of emulsion and foam and may be referred to as

‘foamed emulsion’. The presence of dispersed oil droplets contradicts the stability of the

Introduction

Sandra Böttcher Technische Universität Berlin

foamed emulsion. In numerous studies the destabilizing effect of dispersed oil droplets on

thin films was studied (Zhang et al. 2003; Langevin 2008; Karakashev and Grozdanova

2012; Simjoo et al. 2013). It was shown that oil droplets ‘bridge’ foam lamellae, which

consequently leads to bubble coalescence by thin film rupture (Denkov 2004). In emulsions

with constant surfactant content, oil droplets should have a moderate size, because at a

small size of the oil droplets a sufficiently higher interfacial area needs to be stabilized by

surfactant and at a large size of the oil droplets bridging of thin film occurs. Stability of

foamed emulsions may also be increased by mixtures of QS and β-LG, with β-LG stabiliz-

ing the oil/water-interface and QS stabilizing the air/water-interface. This stabilization

occurs because viscoelastic shear and dilational moduli of β-LG are not as prone to a re-

duction caused by changes in the polarity of the non-aqueous phase. Due to the presence of

negatively charged constituents in the QS extract (Maier et al. 2015a; Tippel et al. 2017)

and the carboxylic group in the molecular structure it is expected that stability of foamed

emulsions increases with increasing pH.

Introduction

Sandra Böttcher Technische Universität Berlin

The aim of this thesis is to analyze and understand interfacial behavior of saponins as well

as their mixture with β-lactoglobulin at aqueous interfaces. In addition, the knowledge on

interfacial properties is applied to explain stabilization and destabilization phenomena in

dispersed model systems like foams and emulsions. Furthermore the gained understanding

of underlying stabilization mechanisms of Quillaja saponins in simple dispersed systems is

used to maximize stability of more complex systems like foamed emulsions.

To accomplish this, the following hypotheses were defined:

1) Saponins from different botanical origin differ in type of aglycone and amount of

linked sugar residues, which affects adsorption and foam properties. It is expected

that triterpenoid saponins have the highest foam stability due to molecular interac-

tions between sugar residues. In addition, it is hypothesized that the type of

aglycone has the highest impact whereas the type and amount of sugar chains has a

minor impact on foam properties. (manuscript I)

2) Similar to β-lactoglobulin (globular protein) Quillaja saponins stabilize foams by

forming a viscoelastic network, which counteracts film rupture and imposed stress.

In mixtures of Quillaja saponins and β-lactoglobulin foam stability may be in-

creased by development of a joint interfacial network, which can withstand

dilational and shear stress. (manuscript II)

3) Interactions between Quillaja saponins and β-lactoglobulin at the oil/water-inter-

face may differ from those at the air/water-interface because oil penetrates between

adsorbed Quillaja saponin molecules and β-lactoglobulin undergoes substantial un-

folding. The interactions may affect not only interfacial rheology but also emulsion

properties. (manuscript III)

4) Quillaja saponins are more efficient in stabilizing the air/water-interface than the

oil/water-interface. To maximize stability of complex dispersed systems (foamed

emulsions) a primary distribution of Quillaja saponins at the air/water-interface

must be ensured. This may be obtained by sequential addition of β-lactoglobulin

and Quillaja saponins to emulsions. It may furthermore be hypothesized that stabil-

ity of the foamed emulsions is maximized at neutral pH due to high electrostatic

repulsion and at moderate oil droplet size. Above and below the moderate oil drop-

let size, stability of the foamed emulsion is reduced due to low amount of free

surfactant and bridging of thin films by oil droplets, respectively. (manuscript IV)

Literature review 33

Sandra Böttcher Technische Universität Berlin

2. Literature review2

2.1. Composition and characterization of saponin extracts

In recent years a lot of effort was made to develop new and improve existing methods for

faster and more reliable determination of saponins. A detailed overview over experimental

protocols reported in recent years was published by Cheok et al. (2014). As for various

plant substances the absence of an easy to fabricate standard, which can be used to quantify

all saponins in an extract complicates matters even more.

For this reason usually fingerprint chromatograms of different saponin extracts are com-

pared to each other (San Martín and Briones 2000). When aiming for identifying and

quantifying unknown saponin molecules, highly sophisticated methods are needed. Various

analytic options were reviewed by Scognamiglio et al. (2015) highlighting 2-D NMR-tech-

niques as the method of choice to determine aglycone structure, sugars and binding sites of

the sugar residues. The author additionally emphasized the role of mass spectrometric (MS)

methods with and without subsequent NMR analysis. In the context of saponin analysis the

use of soft ionization methods like ESI is favorable because of the fragmentation of mole-

cules originating from hard ionization. Less common ionization methods include fast atom

bombardment (FAB) and matrix-assisted laser desorption ionization (MALDI). Masses are

typically determined with time of flight (TOF) analyzers.

Before structural elucidation, saponin extracts are separated into single compounds by

high performance liquid chromatography (HPLC) (San Martín and Briones 2000). For

qualitative analysis the HPLC column is connected to subsequent mass spectrometry (MS).

Various analytic options were reviewed by Scognamiglio et al. (2015) highlighting 2-D

NMR-techniques as the method of choice to determine aglycone structure, sugars and bind-

ing sites of the sugar residues. The author additionally emphasized the role of mass

spectrometric methods with and without subsequent NMR analysis. In recent past, high-

performance thin-layer chromatography (HPTLC) coupled with MALDI-TOF-MS was

used to qualitatively characterize a Quillaja saponin extract (Tippel et al. 2016a). Phenolic

impurities in a Quillaja saponins extract were determined by HPLC-PDA−MSn and NMR

spectroscopy (Maier et al. 2015). Until now a huge variety of saponin derivates were found

in Quillaja saponin extracts: ranging from a molecular weight of 930 to 2,322 g/mol

(Thalhamer and Himmelsbach 2014; Tippel et al. 2016a).

2 Parts of the literature review were published as Manuscript V

Literature review

Sandra Böttcher Technische Universität Berlin

In general, saponins consist of a hydrophobic aglycone structure with linked hydrophilic

sugar residues. Most classifications of saponins either focus on differences in aglycone

structure or amount of linked sugar residues. Two general aglycone structures occur: most

saponins have either a triterpenoid or steroidal aglycone. In addition, various subcategories

of triterpenoid and steroidal aglycone structures exist. For example, triterpenoid saponins

may be oleanane or dammarane type and steroidal saponins may be furostanol or spirosta-

nol type. For further information on structural differences between the mentioned aglycone

structures please refer to Figure 1.

Figure 1 Molecular structure of triterpenoid saponins with A) one sugar chain (monodesmosidic)

or B) two sugar chains (bidesmosidic) with oleanane aglycone structure, C) one or two

linked sugar residues (Mono- and bidesmosidic) with dammarane aglycone structure and

steroid saponins of D) spirostanol and (E) furastanol type. Reproduced from Golemanov

et al. 2013 with permission from The Royal Society of Chemistry.

When classifying saponins based on the amount of sugar residues: monodesmosidic,

bidesmosidic or even tridesmosidic (very uncommon) saponins are known with one, two

and tree sugar chains, respectively. The sugar chains of monodesmosidic saponins are usu-

ally attached at C-3 and the additional sugar chain of bidesmosidic saponins is normally

attached at C-28 of the aglycone (Güçlü-Üstündağ and Mazza 2007). Saponins may also

have acetylated groups like the fatty acyl group in some Quillaja saponins (Hänsel and

Sticher 2010).

The general molecular structure of Quillaja saponins is illustrated in Figure 2.

Literature review 35

Sandra Böttcher Technische Universität Berlin

Figure 2 Molecular structure of bidesmosidic saponins extracted from Quillaja saponaria Molina

with R1-3 representing H, acetyl groups or sugar residues. Adapted from Nord and Kenne

2000 with permission from Elsevier.

There are commercially available saponin extracts from Quillaja saponaria Molina (San

Martín and Briones 2000), Yucca schidigera (Sastre et al. 2016) and Saponaria officinalis

(Oakenfull 1986). Quillaja saponins are the only saponin extracts to date, which are ap-

proved for application in food products in the EU (E 999) and have GRAS status in the US.

There is a whole range of commercial Quillaja saponins extracts available. These extracts

distinctly vary in purity, which ranges between 20 % (Resnik 2004) and >97 %

(Wojciechowski et al. 2016a) and may contain various impurities like phenols, fats, tan-

nins, proteins and sugars (Kuznesof and Soares 2005; Maier et al. 2015a; Tippel et al.

2017).

Literature review

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2.2. Self-assembly and micellar structure

Saponins are well soluble in aqueous solutions and when dissolved in aqueous solutions,

the hydrophilic sugar residues are extensively hydrated. According to Sarnthein-Graf and

La Mesa (2004) 30 water molecules hydrate one QS molecule and Wojciechowski et al.

(2014a) reported 60 water molecules per QS molecule. Saponins are surface active and

therefore adsorb at aqueous interfaces. When the interface is saturated with saponin mole-

cules, micelles are formed in the bulk. Micellar properties may be affected by changes in

pH, ionic strength and temperature (Mitra and Dungan 1997).

The concentration above, which micelles are formed is called ‘critical micelle concen-

tration’ and may be obtained from a plot of the interfacial tension vs. concentration. The

critical micelle concentration is very different between various saponins and saponin ex-

tracts. For example the critical micelle concentration for different Quillaja saponins ranged

from 0.013 g/L (Wojciechowski 2013) up to 0.7 g/L (Mitra and Dungan 1997). Quillaja

saponins can form spherical micelles in aqueous solutions with a diameter of 7.5 nm (Tip-

pel et al. 2016a) (see Figure 3A) and can incorporate water-insoluble compounds. Quillaja

saponins can also form micelles with various substances e.g. lutein esters (Tippel et al.

2016b), cholesterol+L-a-phosphatidylcholine (Demana et al. 2004). With about 130 nm,

Quillaja saponin micelles loaded with lutein esters are distinctively larger compared to un-

loaded micelles. It is possible that elongated/worm-like micelles are formed between

micelles consisting of QS and hydrophilic substances at low pH, see Figure 3B. The elon-

gation was attributed to a decrease in electrostatic repulsion of the charged head-groups

and thus, an increase in the critical packing parameter, which is responsible for the elonga-

tion of the micelles (Tippel et al. 2016a).

Figure 3 Cryo-TEM micrographs of Quillaja saponin/lutein ester micelles in (A) aqueous unbuff-

ered solution (spherical) and B) in buffer at pH 3 (elongated). Reproduced from Tippel et

al. 2016a with permission from Elsevier.

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2.3. Linking molecular structure and behavior at aqueous interfaces

When trying to link molecular structure with interfacial behavior it has to be kept in mind,

that extracts are prone to differences in saponin content and composition. A detailed col-

lection of all reported saponins would be beyond the scope of this literature review. There

are excellent reviews on the molecular structure of saponins found in different plants (Sparg

et al. 2004; Vincken et al. 2007; Dinda et al. 2010). Usually saponin extracts also contain

numerous plant residues, which may affect interfacial properties as shown by Pagureva et

al. (2016). Figure 4 provides an overview over classification of the discussed saponins in

this work with respect to aglycone structure and amount of sugar residues.

Figure 4 Structural classification of discussed saponin extracts in this thesis with respect to

A) aglycone structure B) amount of sugar residues

Saponins

Triterpenoid Steroid

Saponins

Mono-

desmosidic

Bi-

desmosidic

Mono- &

Bidesmosidic

Quillaja saponaria

Molina (QS)

Camellia oleifera

Abel (TS)

Aesculus hippo-

castanum (ESC)

Glycyrrhiza glabra (GA)

Gypsophia species (GYP)

Sapindus mukurossi

(BSC)

Yucca schidigera

Tribulus terrestris (TT)

Oleanane

type

Ursolic &

Oleanane type

Ilex paraguariensis

Quillaja saponaria

Molina (QS)

Gypsophia species

(GYP)

Panax ginseng (GS)

Camellia oleifera

Abel (TS)

Aesculus hippo-

castanum (ESC)

Glycyrrhiza glabra

(GA)

Tribulus terrestris

(TT)

Sapindus mukurossi

(BSC)

Yucca schidigera

Dammarane

type

Panax ginseng (GS)

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2.3.1. Interfacial configuration of saponins at the air/water-interface

For the determination of structure-function-relationship, information on the orientation and

interfacial arrangement of the adsorbed molecules are needed. The adsorbed surfactant

layer can be described with the equation of state and adsorption isotherm. The former equa-

tion describes the relationship of the surface pressure/surface tension as a function of

surfactant concentration at the interface. In contrast the adsorption isotherm characterizes

the dependence of the adsorbed amount of surfactant on the bulk concentration. There were

different types of models used in studies on saponins so far, like the Langmuir or Volmer,

which can be fitted to the experimental data. The Langmuir model (Eq. 1a+b) was derived

for localized adsorption, which means that every molecule has a defined site at the interface

and cannot freely diffuse in the interfacial layer.

Langmuir a) adsorption isotherm and b) equation of state

(1a) KcS= Γ

Γ∞−Γ

(1b) σ= σ0+ k𝐵𝐵TΓ∞ln (1 −Γ/Γ∞)

where K is the adsorption parameter, c0 the surfactant concentration in the bulk, Γ the sur-

face concentration, 𝛤𝛤∞- maximum possible value of surface concentration at c0→∞, kB the

Boltzmann constant, T the absolute temperature, σ the surface tension and σ0 surface tension

of the pure solvent.

The Langmuir model can be expanded considering interactions between the molecules,

which results in the Frumkin model (Eq. 2a+b).

Frumkin a) adsorption isotherm and b) equation of state

(2a) Kc0= Γ

Γ∞−Γexp �−2βΓ

kT�

(2b) σ= σ0+kTΓ∞ln �1−Γ

Γ∞�+βΓ²

where 𝛽𝛽 is the interaction parameter.

The Volmer (Eq. 3a+b) and van-der-Waals (Eq. 4a+b) model describe non-localized ad-

sorption, with the assumption that molecules do not occupy a particular site and can freely

diffuse in interfacial layer, where the latter takes molecular interactions into account (Kolev

et al. 2002).

Volmer a) adsorption isotherm and b) equation of state

(3a) Kc0= Γ

Γ∞−Γexp �Γ

Γ∞−Γ�

(3b) σ= σ0+k𝐵𝐵TΓ∞Γ

Γ∞−Γ

Literature review 39

Sandra Böttcher Technische Universität Berlin

Van der Waals a) adsorption isotherm and b) equation of state

(4a) Kc0= Γ

Γ∞−Γexp �Γ

Γ∞−Γ−2βΓ

k𝐵𝐵T�

(4b) σ= σ0+k𝐵𝐵TΓ∞Γ

Γ∞−Γ+βΓ²

From the fitting of the interfacial tension data the area per molecule can be obtained. Com-

bining the area per molecule with data on the molecular dimension, allows drawing

conclusions on the orientation of the molecules at the interface.

In case of saponins, an interfacial area smaller than 0.75 nm² is related to the orientation

of the molecules in the end-on/side-on configuration and over 0.75 nm in the lay-on con-

figuration (Pagureva et al. 2016). In the side-on configuration the aglycones of neighboring

monodesmosidic saponin molecules are arranged parallel to each other in the hydrophobic

phase and perpendicular to the interface (see Figure 5A). The end-on configuration is sim-

ilar to the side-on configuration but describes the orientation of bidesmosidic saponins

where one sugar residue is additionally facing into the hydrophobic phase besides the agly-

cone (see Figure 5B). Bidesmosidic saponins can also arrange in the lay-on configuration

where both sugar chains are facing towards the water phase, leaving the aglycone orientated

parallel to the interface (see Figure 5C).

Monodesmosidic saponins usually occupy an area of around 0.3 nm², which corresponds

to the end-on configuration. Bidesmosidic triterpenoid saponins are more likely to arrange

in a lay-on configuration, which corresponds to an interfacial area per molecule of 1 nm²

(Stanimirova et al. 2011; Pagureva et al. 2016). However, it remains unclear if Quillaja

saponins from different extracts always arrange in the lay-on configuration at the interface.

In the past interfacial tension isotherms of various Quillaja saponin extracts were analyzed

and data of 0.3 to 1 nm² for the area per molecule were reported by different research groups

(Stanimirova et al. 2011; Kezwon and Wojciechowski 2014). The differences in area per

molecule obtained at the air/water-interface were explained by the use of different Quillaja

saponin extracts.

An exception is the extract from Panax ginseng (GS) with bidesmosidic saponins, which

had an interfacial area per molecule of around 0.5 nm² (Pagureva et al. 2016). The end-on

configuration observed for this bidesmosidic saponin may be justified because of the short

sugar chains attached to the saponin, which make this configuration more advantageous.

Steroidal saponins are often a mixture of mono- and bidesmosidic saponins and therefore

interfacial area is between 0.3 and 1 nm² (Pagureva et al. 2016).

Literature review

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Figure 5 Interfacial configuration of saponins A) end-on configuration, B) side-on configuration

and C) lay-on configuration. Blue parts of the schematic molecules indicate hydrophilic

parts (sugar residues) and brown parts represent hydrophobic parts (aglycone). Adapted

from Golemanov et al. 2013 with permission from The Royal Society of Chemistry.

In several studies (Pagureva et al. 2016; Golemanov et al. 2014; Golemanov et al. 2013)

where 13 different saponins from different botanical origins were compared, a group of

saponins with high dilational and shear viscoelastic properties was identified. This group

consists of extracts from the plants Camellia oleifera Abel (TS), Aesculus hippocastanum

(ESC), Panax ginseng (GS), Sapindus mukurossi (BSC) and Quillaja saponaria Molina

(QS). All named saponins have an oleanane type aglycone but GS, which has a dammarane

aglycone. Furthermore TS and ESC can be classified as monodesmosidic saponins and GS

and QS as bidesmosidic saponins. BSC consists mainly of monodesmosidic saponins and

a small portion of bidesmosidic saponins.

Pagureva et al. (2016) have shown using the van-der-Waals adsorption isotherm that

some interfacial layers of monodesmosidic saponins (here: TS and ESC) and bidesmosidic

saponin with short sugar chains (here: GS) can undergo phase transition, which means that

the interaction parameter derived from the van der Waals isotherm is greater than 3.375.

Phase transition was attributed on the one hand to strong hydrophobic interactions between

aglycones and on the other hand on the hydrogen bonds between sugar residues. The ob-

served phase transition seems to lead to high dilational and shear elasticity as well as high

interfacial viscosity for these saponins (TS, ESC and GS). When subjecting the interfacial

layers to increasing surface deformation a steep decline of the dilational elasticity was ob-

served. Although the elastic modulus is still very high at increased amplitudes, it has to be

Literature review 41

Sandra Böttcher Technische Universität Berlin

noted that intermolecular bonds of TS, ESC and GS are more fragile when dilational force

is applied than for instance QS and BSC, which are less prone to reduction of dilational

viscoelasticity imposed by surface deformation. High dilational and shear viscoelasticity is

associated with intermolecular interactions (here: hydrogen bonds). The formation of hy-

drogen bonds was indirectly shown by the addition of the chaotropic reagent urea to an

interfacial layer of TS. As a chaotropic agent, urea unlinks intermolecular hydrogen bonds

thus, leading to a total loss of viscoelastic properties (Golemanov et al. 2014).

In contrast, until now all examined steroidal saponins (from Yucca Schidigera, Tribulus

terrestris and Trigonella foenum-graecum) neither showed dilational nor shear elasticity or

significant surface viscosity (Golemanov et al. 2013; Pagureva et al. 2016). It is supposed

that interfacial layers are in a rather liquid condensed state with only weak intermolecular

interactions.

The very small saponin glycyrrhizin from the root of Glycyrrhiza glabra was also not

able to form a viscoelastic film at the air/water-interface, which was attributed to the low

solubility because of only few hydrophilic sugar residues in the molecular structure

(Golemanov et al. 2013). In addition, the amount of carboxylic groups is relatively high

with respect to the size of the molecule in comparison to other saponins. The charged car-

boxylic groups may cause repulsion inside the interfacial layer and therefore prevent

intermolecular interactions, which may lead to a viscoelastic film.

2.3.2. Supramolecular interfacial configuration of Quillaja saponins at the air/water-

interface

Golemanov et al. (2012) analyzed the behavior (relaxation times) of a Quillaja saponin

layer under shear and dilational stress and derived a reasonable model for the interfacial

packaging of Quillaja saponin molecules. The authors hypothesized that molecules are

packed in domains and strong intermolecular interactions exists between saponin molecules

inside the domain as indicated by high dilational and shear moduli. Inside a domain, Quil-

laja saponin molecules orientate themselves similarly and exhibit strong intermolecular

interactions (see Figure 6). At the boundaries of two neighboring domains weaker interac-

tions and higher free energy occurs because boundary molecules of different domains may

have divergent orientation.

Literature review

Sandra Böttcher Technische Universität Berlin

Figure 6 Supramolecular domain structure of Quillaja saponins at the air/water-interface. A) Inside

a domain: Quillaja saponins (I) orientate themselves in the same direction, boundary mol-

ecules can have different orientations B) Neighboring domains can have different

orientation of inside molecules. Adapted from Golemanov et al. 2012 with permission

from The Royal Society of Chemistry.

When shear stress is applied Quillaja saponin molecules orientate in the direction of the

applied stress and domain boundaries lengthen thereby increasing the energy of the inter-

facial layer (see Figure 7). Up to a certain level of shear stress the interfacial deformation

is reversible and saponin molecules realign fast after the end of the shear stress (elastic

response). When shear stress becomes too high, the domains laterally shift in the direction

of shear stress, which is irreversible and results in a no longer elastic but viscous response.

When the interfacial layer is subjected to expansion it is expected that the boundary region

between the domains widen since these regions have relatively weak intermolecular bonds.

Quillaja saponin molecules can diffuse from the subsurface to the widened boundary re-

gions with subsequent re-orientation at the interface. Based on the differing interfacial

configuration and interfacial rheology one has to assume that interfacial structure of Quil-

laja saponins is different from monodesmosidic saponins. Until now, there has been no

proposed model on the supramolecular structure of monodesmosidic saponins.

Figure 7 Elongation of the Quillaja saponin domains when shear stress is applied. Reproduced

from Golemanov et al. 2012 with permission from The Royal Society of Chemistry.

Literature review 43

Sandra Böttcher Technische Universität Berlin

2.3.3. Impact of the hydrophobic phase on interfacial properties of various saponins

Oil as a hydrophobic phase can influence interfacial properties by (1) penetrating between

adsorbed surfactant molecules and (2) by solvation of surfactant molecules in the hydro-

phobic phase, thus reducing interfacial concentration and surface pressure. The first

mechanism usually applies for tightly anchored surfactants and proteins that are non-solu-

ble in the hydrophobic phase and the second mechanism for surfactants soluble in the

hydrophobic phase like non-ionic low-molecular weight surfactants. The first mechanism

applies for most saponins but for TS and BSC considerable solubility in the hydrophobic

phase was reported. It is possible that solubility of TS and BSC in the hydrophobic phase

additionally contributes to the reduction in viscoelasticity (Golemanov et al. 2014).

Triglycerides (trycaprylin and olive oil) tend to reduce dilational and shear viscoelastic-

ity to a higher extend than linear alkanes (hexane or tetradecane). Golemanov et al. (2014)

explained this with the bulky size of the triglyceride molecules and the tendency of their

hydrophilic head groups to get in contact with the aqueous phase. As Figure 8 shows

monodesmosidic saponins orientate differently at the interface in comparison to bidesmo-

sidic saponins and are more affected by changes in the hydrophobic phase.

Figure 8 Orientation of monodesmosidic saponins at the (A) air (B) hexadecane (representing lin-

ear alkanes) and (C) Tricaprylin (representing triglycerides) as well as bidesmosidic

saponins at (D) at different interfaces. Blue parts of the schematic molecules indicate

hydrophilic parts (sugar residues), brown parts represent hydrophilic parts (aglycone)

and dark blue lines show intermolecular hydrogen bonds. Adapted from Ref.

(Golemanov et al. 2014) with permission from The Royal Society of Chemistry.

Literature review

Sandra Böttcher Technische Universität Berlin

2.4. Interactions of saponins with other (food) components

Interactions of proteins and surfactants can result in deviating interfacial properties of mix-

tures (Lech et al. 2014; Mackie and Wilde 2005; Dan et al. 2013; Maldonado-Valderrama

and Patino 2010). It is well know that low-molecular weight surfactants like SDS can in-

teract with proteins (Pradines et al. 2009; Ulaganathan et al. 2012; Lech et al. 2014). As a

food additive, interactions between Quillaja saponins and food proteins are relevant.

In the past, various studies examined the interactions of Quillaja saponin with food com-

ponents like β-lactoglobulin (Kezwon and Wojciechowski 2014; Piotrowski et al. 2012),

β-casein (Kezwon and Wojciechowski 2014; Wojciechowski et al. 2014b), lysozyme (Kez-

won and Wojciechowski 2014; Wojciechowski et al. 2011) and gelatin (Sarnthein-Graf and

La Mesa 2004).

Studying model Quillaja saponin/food components-systems is crucial in order to fore-

cast interactions in real food products. Many publications determined protein-surfactant

interactions, which is a topic far from trivial (Dan et al. 2013; Maldonado-Valderrama and

Patino 2010; Krägel et al. 2008). Especially, protein-Quillaja saponin-systems are tremen-

dously different from protein-surfactant-systems because of the unique interfacial

properties of Quillaja saponins. To summarize previously mentioned studies, evidence for

complex formation between Quillaja saponins and the random coil milk protein β-casein,

globular protein β-lactoglobulin (β-LG) and hen egg lysozyme were found. It has to be

noted that most assumptions on complex-formation were based on interfacial tension, in-

terfacial rheology and fluorescence measurements.

By far most studies evaluated the interactions between QS and β-LG as well at the

air/water (Piotrowski et al. 2012) and tetradecane/water-interface (Piotrowski et al. 2012).

In contrast to β-LG, lysozyme is positively charged, which enables electrostatic attraction

to Quillaja saponins. But, it hast to be kept in mind that the negative charge of QS is only

weak since only one carboxylic group is present in Quillaja saponins. In a different study

on QS/gelatin interaction only minor interactions between QS and gelatin were found

(Sarnthein-Graf and La Mesa 2004). In fact, in mixtures of QS/lysozyme adsorption rate in

dynamic interfacial tension experiments at the air/water-interface increased compared to

only QS. This increased adsorption rate was attributed to interfacial complex formation

through electrostatic and hydrophobic interactions and additionally through sugar binding

sites (Kezwon and Wojciechowski 2014).

Literature review 45

Sandra Böttcher Technische Universität Berlin

When evaluating β-casein/QS interactions at various interfaces it has to be noted that β-

casein is a random coil and not a globular protein like β-LG and lysozyme. As a conse-

quence, no tertiary structures like a hydrophobic calyx are present in β-casein. At low QS/β-

casein concentrations, complex formation led to increased adsorption rate because the hy-

drophilic character was increased by electrostatic interaction of one or two QS molecules

with the positively charged N-terminus of β-casein. At higher QS/β-casein concentrations

this effect was diminished and interfacial activity was reduced. This effect was attributed

to additional QS molecules, which electrostatically interact with other positively charged

parts of β-casein leading to reduced surface active properties. The described observations

were valid for the air, tetradecane and olive-oil/water-interface (Wojciechowski et al.

2014b).

Another group focused their research on the miscibility of Quillaja in the presence of

Na-caseinate, pea protein and different lecithins under heat and pH influence (Reichert et

al. 2015; Reichert et al. 2016). As expected heat and pH affected miscibility in mixed sys-

tems tremendously. Mixtures of QS with Na-caseinate were miscible at pH 7 and different

concentration ratios but when subjected to heat and reduction of the pH, sufficient aggre-

gation was observed. Aggregation behavior because of pH reduction was on the one hand

attributed to the self-aggregation of casein at the isoelectric point and due to electrostatic

interactions at lower pH because of the opposite charge of both molecules. Aggregation of

the QS/Na-caseinate mixture was associated with the behavior of each substance upon heat-

ing (turbidity increase of QS, phase transition of αs-casein or unfolding of β-casein) and

possible hydrophobic interactions. It was further emphasized that impurities like phenols

can also contribute to the aggregation behavior by forming protein-phenol-interactions.

The literature review showed that saponins are a diverse class of natural emulsifiers and

that they may use to stabilize dispersed systems. In addition, the interaction between sapo-

nins and food proteins is highly relevant when applying saponins in food systems.

Manuscript I

Sandra Böttcher Technische Universität Berlin

Manuscript I

1 Interfacial Properties of Saponin Extracts

and Their Impact on Foam Characteristics

Food Biophysics, 2016, V. 11, No. 1, pp 91–100.

The final publication is available at Springer via http://dx.doi.org/10.1007/s11483-015-

9420-5.

Authors

Sandra Böttchera

Stephan Druscha

a Technische Universität Berlin, Institute for Food Technology and Food Chemistry

Department of Food Technology and Food Material Science

Königin-Luise-Str.22, 14195 Berlin

Manuscript I

Sandra Böttcher Technische Universität Berlin

I-1 Abstract

Saponins from various botanical origins highly differ in molecular structure. Little is known

of the influence of structural differences between the different saponins on interfacial ten-

sion, short-term adsorption and foam properties at the air-water interface (a/w). In this study

five triterpenoid saponins, with three of these being monodesmosidic and two bidesmosidic

as well as one steroid saponin, were analyzed. Interfacial tension isotherms were measured

using a tensiometer with a Wilhelmy plate and were fitted using the modified Frumkin

model. For characterization of the short-term adsorption at the a/w-interface, two-fluid nee-

dle experiments were performed. Foaming, foam stability and foam structure were analyzed

using a foaming device. A new method for semi-quantitative analysis of different foam

structures was established. In addition, the impact of pH and ionic strength (addition of

NaCl) on interfacial tension and foam properties were determined. The short-term adsorp-

tion of all saponins was limited by an additional barrier and was not diffusion-limited.

Extracts from Quillaja saponaria Molina (QS), Gypsophila (GYP), Camellia oleifera Abel

(TS) and Aesculus hippocastanum (ESC) lowered the interfacial tension to 37-42 mN/m

and produced stable foams. The steroid saponin from Tribulus terrestris (TT) and the

monodesmosidic saponin from Glycyrrhiza glabra (GA) had only poor interfacial and foam

properties. Foams made from QS and GYP were only little affected by changes in pH and

ionic strength. A reduction of the pH from 5 to 3 increased stability of foams made from

GA significantly. Foams made from ESC and TS were negatively affected by increasing

ionic strength.

Manuscript I

Sandra Böttcher Technische Universität Berlin

I-2 Introduction

Secondary plant metabolites like e.g. saponins are nowadays frequently standardized and

used for nutritional or medical reasons in human nutrition. There has been an overwhelming

amount of research on health related aspects of plant-derived food ingredients throughout

the last two decades (Raskin et al. 2002; Schroeter et al. 2010; Pojer et al. 2013). In contrast

scientific literature contributing to our understanding of plant extracts in foods and their

technological functionality is scarce.

Saponins are a heterogonous amphiphilic class of substances, which consist of a hydro-

phobic aglycone and hydrophilic sugar residues. There are comprehensive studies on the

distribution and structure of saponins in a variety of botanical sources (Haralampidis et al.

2002; Sparg et al. 2004; Vincken et al. 2007; Dinda et al. 2010). Numerous variations in

the aglycone structure as well as the amount, type and location of the sugar residues are

known until now. The quantitative determination of the various saponin types in a botanical

extract remains a field of ongoing research, which requires specialized equipment, like LC-

MS or HPTLC and which is limited by the availability of standard substances (Oleszek and

Bialy 2006; Yao et al. 2008).

Due to the above mentioned structural properties of saponins from a technological point

of view, research focused on the interfacial activity and their use in food applications (Kez-

won and Wojciechowski 2014). The wide range of the botanical origin results in extracts

with different saponin mixtures, which makes it challenging to identify structure-function

relationships. Even saponin extracts from the same botanical origin may exhibit different

interfacial properties (Mitra and Dungan 1997). Despite these aspects, the interest in sapo-

nins increases because saponins are natural surface active molecules and some saponin

extracts exhibit a high interfacial activity. Saponins from the bark of Quillaja saponaria

Molina are generally recognized as safe according to the USDA and they are approved for

application in beverages in the EU as foam stabilizer.

Different studies analyzed basic aspects of the interfacial properties and orientation of

the molecules of various Quillaja extracts at an interface (Stanimirova et al. 2011;

Golemanov et al. 2012; Wojciechowski 2013). Quillaja saponins have a high molecular

weight because of their fatty acyl and two sugar moieties, which influence the orienta-

tion/configuration (Golemanov et al. 2012). Recently, Yang et al. (2013) published data on

the influence of temperature, pH change and ionic strength on the stability of emulsions

containing Quillaja saponins. Apart from the research on Quillaja saponins, Golemanov et

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al. (2013) published data on the surface elasticity and viscosity of 12 triterpenoid and ster-

oid saponins. The authors observed a high elasticity and viscosity of the surface layers for

triterpenoid saponins. In contrast, steroid saponins exhibited poor surface elasticity. With

respect to food applications, only in a very limited number of studies specific aspects of the

foam properties have been investigated (Chen et al. 2010; Wojciechowski et al. 2011; Pi-

otrowski et al. 2012). To the best of our knowledge until now there has been no

comprehensive approach on the characterization of saponin-based foams considering foam-

ing, foam stability and foam structure for a range of saponin extracts from different

botanical origin.

Aim of the present study was to collect data on the basic interfacial properties of saponin

extracts from various botanical origins with their foam properties to contribute to our un-

derstanding how these parameters are interconnected. This study is a preliminary

investigation, which requires more-in-depth mechanistic studies with chemically well-de-

fined saponin extracts. To fulfill this aim the interfacial tension isotherms of saponin

extracts were determined, fitted with the Frumkin model and the critical micelle concentra-

tion was calculated. For general characterization of the saponin extracts the foaming

properties, interfacial tension and short-term adsorption at the air/water interface at pH 5

were determined. In a second step, the ionic properties of the saponins were determined

and the influence of a pH reduction from 5 to 3 and increase of ionic strength from 0 mM

to 100 and 500 mM on the foaming properties and interfacial tension was evaluated.

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I-3 Materials and Methods

Six different saponin extracts were included in the present study based on their molecular

structure, see Table I-1. The saponins comprised five triterpenoid and one steroidal saponin.

Two of the triterpenoid saponins were bidesmosidic and the remaining three monodesmo-

sidic. As indicated from the literature, saponins may additionally vary in molecular weight

and the number of carboxylic groups.

Table I-1 Origin, purity and general chemical properties of the six saponin extracts

Botanical

origin Saponin Abbre-

vation

Plant

material

Saponin

concen-

tration*

[%]

Aglycone Sugar resi-

dues

Molecular

weight

[g/mol]

Quillaja sapo-

naria Molina

Quillaja QS Bark 69.2 Triterpenoid Bidesmosidic

1400-

2300

Gypsophila Gypsophila GYP n.s. 45.5** Triterpenoid Bidesmosidic

1400-

1700

Camellia oleif-

era Abel

Tea Saponin TS Seeds 95.3 Triterpenoid

Monodesmo-

sidic

1200-

1300

Aesculus hip-

pocastanum

Escin ESC n.s. 99.1 Triterpenoid

Monodesmo-

sidic

11004

Glycyrrhiza

glabra

Glycyrrhizic

Acid Am-

monium Salt

GA Root 95.1 Triterpenoid Monodesmo-

sidic 839

Tribulus ter-

restris

Tribulus ter-

restris

TT Fruits 90.4 Steroid

Mono- and

bidesmosidic

600-11005

* calculated on dry matter, ** determined by vanillin-sulphuric acid assay, n.s.- not specified

1 Bankefors et al. 2008; Thalhamer and Himmelsbach 2014

2 Frechet et al. 1991; Chen et al. 2011; Yao et al. 2011; Voutquenne-Nazabadioko et al. 2013;

Pertuit et al. 2014

3 Huang et al. 2007; Kuo et al. 2010; Zhang et al. 2012; Zhou et al. 2014

4 Wulff and Tschesche 1969

5 Dinchev et al. 2008

An extract from the bark of Quillaja saponaria Molina (QS) with a specified saponin con-

tent of 69.2 % (dry matter) was provided by Ingredion Germany GmbH (Hamburg,

Germany). Gyphsophila extract (GYP) was a kind gift of Dr. H. Schmittmann GmbH (Vel-

bert, Germany). The saponin content as determined by the vanillin-sulphuric acid method

amounted to 45.5 % (dry matter). A saponin extract from the seed of Camellia oleifera

Abel (TS) with a saponin content of 95.1 % (dry matter) was provided by Changsha Nulant

Chem. Co., Ltd. (Changsha City, Hunan, China) . The extract of the fruits of Tribulus ter-

restris (TT) was kindly donated by Xi'An Union Pharmpro Co., Ltd (Xi’An, Shaanxi,

China) and its saponin content was specified with 90.4 % (dry matter). Saponin-rich extract

from Aesculus hippocastanum (ESC), 99.1 % (dry matter) and Glycyrrhiza glabra (GA),

95.1 % (dry matter) as well as the ionic surfactant sodium dodecyl sulfate (SDS), >99 %

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were purchased from Sigma Aldrich Chemie GmbH (Steinheim, Germany). For all exper-

iments mono-distilled water was used. Solutions made of GA were heated to 40°C to solve

the surfactant. Vanillin (100 %), ethanol (99.5 %) and sulphuric acid (96 %) were pur-

chased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany).

No specific data on the quantitative composition of the saponin extracts were provided by

the suppliers, but a range of literature dealing with the composition of the saponin fraction

in different plants is available. In general, saponins of one botanical species only have slight

differences in molecular structure. GA has a specific molecular structure and is not a mix-

ture of various saponins. For QS, TT, TS and ESC the specific botanical origin is provided

by the supplier, but not for GYP. A range of different studies identified common saponins

in QS, TT, TS, ESC and GYP. General structural properties and the average molecular

weight required to model the adsorption isotherms and to calculate the hydrodynamic radii

were derived from the literature, see Table I-1.

I-3.1 Fourier transform infrared spectroscopy (FTIR)

For the analysis of the presence of different chemical bonds in the six different saponin

extracts, the FTIR-spectra of all saponins were measured using a Bruker Tensor 27 (Bruker,

Germany) with a diamond ATR. Prior to the measurements of the samples the instrument

was calibrated against air. Spectra between 4000-400 cm-1 were obtained using 32 scans

with a resolution of 4 cm-1. Results were normalized to compare different spectra. A dried

sample of the QS extract was used for the FTIR measurement. For the stretch of C-OH

groups a broad peak around 3400 cm-1 occurs in the spectra. Peaks between 3200 and 2800

cm-1 represent the stretching of sp2 and sp3-C-H bonds and C═C bonds can be identified at

a wavenumber of around 1600 cm-1. Most important in this study is the stretch of the car-

bonyl bonds (C═O) at 1730-1700 cm-1. If there is no peak in this region, it can be concluded

that no carboxylic groups are present in the saponin extract.

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I-3.2 Conductivity measurements

To determine the ionic character of the different saponin extracts, a dilution test was con-

ducted. The conductivity was measured with the Seven Easy by Mettler Toledo and an

InLab 731 electrode. An aliquot of the diluted saponin extract was stirred and the conduc-

tivity was measured. Then a part of the sampled was removed and replaced with distilled

water. Again the conductivity was recorded and the relation between conductivity and con-

centration was plotted. All dilution steps were equidistant. The dilution test is an option for

fast determination of the CMC of ionic surfactants (Khan and Shah 2008). In solutions of

non-ionic surfactants there is a linear dependency between the conductivity and the con-

centration of a substance. In contrast, in ionic extracts there is a breaking point at the CMC

(=CMCcond) with the slope of the curve being more flat above the CMC and more steep

below the CMC. Above the breaking point the charged parts of the molecule are shielded

because of micelle formation.

I-3.3 Interfacial tension measurements using Wilhelmy plate

The interfacial tension of the six different extracts was determined using a K11 tensiometer

equipped with a Wilhelmy plate (KRÜSS GmbH, Hamburg, Germany). An aliquot of 80

mL of the diluted extracts was equilibrated to 20±1°C and placed into the measurement

glass vessel. Prior to the analysis the Wilhelmy plate was thoroughly rinsed with distilled

water and acetone. Then the plate was heated above a lab burner to remove possible con-

taminants. The plate was cooled down and then again rinsed with distilled water. Finally

the plate was wetted with the diluted extract for two times and then immersed with a speed

of 100 mm/min into the solution before it reached its final position at the interface. The

interfacial tension was measured for a period of 400 s. The vessel was equipped with a

jacket to ensure a constant temperature throughout the measurement. Air circulation was

eliminated by a plastic shield.

To determine the critical micelle concentration (CMC) a series of different concentra-

tions of the extracts ranging from 0.0001 to 2 %wt were analyzed. The CMC was obtained

by calculating the intersection between the flat curve above and the steep curve below the

break in the interfacial tension isotherm. The interfacial tension isotherms were fitted with

the modified Frumkin model using the Software “IsoFit”.

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The following two equations describe the equation of state (Eq.I-1) and adsorption isotherm

(Eq.I-2) of the modified Frumkin model:

−𝜋𝜋𝜋𝜋

𝑅𝑅𝑅𝑅 =ln(1−𝜃𝜃)+𝑎𝑎𝜃𝜃2 (I-1)

𝑏𝑏𝑏𝑏=𝜃𝜃

1−𝜃𝜃exp (−2𝑎𝑎𝜃𝜃) (I-2)

where Π is the surface pressure, ω the molar surface area, R the general gas constant, T the

absolute temperature, θ the surface coverage, a the interaction parameter, b the adsorption

equilibrium constant and c the bulk concentration. The Frumkin model assumes a mono-

layer of the surfactant at the interface and extends the Langmuir model by considering

interactions between the molecules. In addition, the modified Frumkin model takes com-

pressibility ε of the monolayer because of increasing surface pressure into account: ω=ω0(1-

εΠ). This changes ω into a dependent parameter. The parameter ω0 is the molar area at

Π=0.

I-3.4 Short-term adsorption of saponin extracts on the air-water interface

For experiments on short-term adsorption the drop shape analysis system OCA-20

(DataPhysics Instruments GmbH, Filderstadt, Germany) was used as described by Tamm

et al. (2012). Briefly, a two-fluid needle composed of a large needle (d=1.65 mm) and a

smaller needle (d=0.51 mm), which was placed inside the large needle, was used for droplet

generation. Through the outer channel (large needle) a droplet of distilled water with a

volume of 12 µL was generated manually. An aliquot of 3 µL of the saponin solution was

dosed with the automatic dosing unit through the small needle inside the water droplet. A

CCD camera recorded a high speed video with 200 fps of the drop shape for a maximum

of 20 s. Interfacial tension was calculated from the shape of the droplet and based on these

data the lag-time, surface pressure after 5 s after the start of the adsorption Π5s and the

experimental diffusion coefficient Dexp were derived. The lag-time is defined as the time

period after the droplet volume is expanded by 1 % until an absolute change of >2.0 mN/m

of the interfacial tension and describes the speed of a molecule to reach the blank surface.

This speed depends on the generated current inside the molecule because of the injection,

as well on diffusion based on concentration gradients.

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The short-term approximation (t→0) for the Ward-Tordai equation (Ward and Tordai 1946)

combined with the Henry Equation (Eq. I-3) was used to calculate Dexp:

𝑑𝑑𝑑𝑑

𝑑𝑑𝑑𝑑1/2=−2𝑅𝑅𝑅𝑅∙𝑏𝑏0�𝐷𝐷𝑒𝑒𝑒𝑒𝑒𝑒

𝜋𝜋 , 𝑡𝑡→0 (I-3)

where σ is the interfacial tension, t is the time and c0 the concentration of the saponin in the

bulk solution. For theoretical values for Dcalc the maximum length of each molecule was

estimated using bond lengths and angles. The maximum length of each molecule was de-

fined as the diameter (2r) of a spherical shape although the saponins have a smaller width

and height. The Stokes-Einstein Equation (Eq. I-4) was used to calculate the theoretical

diffusion coefficient Dcalc:

𝐷𝐷𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 =𝑘𝑘𝐵𝐵∙𝑅𝑅

6𝜋𝜋∙𝜂𝜂∙𝑟𝑟 (I-4)

where kB is the Boltzmann constant, η is the dynamic viscosity and r is the hydrodynamic

radius a the spherical molecule. The values for Dexp were compared to theoretical values

Dcalc. If experimental values for the diffusion coefficient are smaller than the calculated

values, the adsorption is not diffusion limited but controlled by a further adsorption barrier.

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I-3.5 Foaming and foam stability

To analyze the foaming properties of the saponin extracts a commercially available foam-

ing device DFA 100 was used (KRÜSS GmbH, Hamburg, Germany) (Lunkenheimer et al.

2010). Instead of using an absolute concentration, which would neglect differences in the

interfacial activity, a multiple of the CMC of the individual extracts was chosen for the

experiments (0.5 and 2-fold). Following parameters were fixed in all experiments: A vol-

ume of 50 mL of the saponin solution was poured in a glass column with a diameter of

40 mm equipped with a porous glass frit with a pore size of 40-100 µm (FL 4502). Pres-

surized air was purged through the frit at a rate of 0.15 L/min and foaming was stopped at

a total height of 180 mm (sum of liquid and foam). Foam generation and stability was mon-

itored for 3600 s and the height of the foam and the remaining liquid as well as a brightness

profile were measured and recorded by transmissibility measurement with a frame rate of

2/s. In addition, every 10 min an image of the foam was taken. To minimize external influ-

ences on the brightness profile and image all experiments were carried out under exclusion

of light. In-between individual measurements the device was thoroughly rinsed with dis-

tilled water. All experiments were carried out at least in duplicate.

For comprehensive foam characterization multiple parameters were evaluated, see Fig-

ure A-2. Foaming was characterized by the foaming speed kf from the start of the foaming

until the maximum foam height (f-max). The remaining foam height fn at time point n char-

acterizes the percentaged amount of foam still present in relation to f-max. Small fn values

indicate high foam decay. The parameters foam density fDen,n and foam density at f-max

fDen,f-max describe the percentage of incorporated liquid in the foam. The drainage is defined

as the percentage of liquid draining from the foam in relation to the liquid height at the f-

max. In dense foams the variation as determined from fivefold replicates, for all parameters

calculated without the liquid height as a variable (like foam stability and foaming parame-

ters) was below 3 % and about 15-20 % for parameters, which were calculated from the

liquid height (like drainage and foam density). In less dense foams the variation for all

parameters calculated without liquid height was between 5-12 % and about 30-40 % for

parameters derived from the liquid height.

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I-3.6 Determination of foam structure with analysis of brightness profiles and foam

pictures

In order to characterize the foams in more detail and to overcome the high variation in

parameters based on liquid height detection, a new method was established. Therefore, the

brightness profile generated by the foaming device was analyzed, see Figure A-3. The

brightness profile records the average transmissibility of the foam and liquid. Each pixel

on the vertical axis displays the average transmissibility at a certain height of measurement

and each pixel on the horizontal axis represents the average transmissibility at a specific

time point. At different time points the brightness distribution within the foam in the col-

umn was measured in an area of 1 px width, which corresponds to a specific time point,

using Adobe Photoshop CS6 Extended. The brightness distribution ranges from 0-255. 0

represents black and 255 represents white. In this study, data points between 0 and 253

were used to eliminate the background color. From the brightness distribution the median

(BDm,n) as well as the width (BDw,n) between the d10 (10 % of the brightness values are

beneath this value) and d90 were calculated for individual time points n. The higher the BDm

is, the more light is transmitted through the foam indicating that the foam is less dense. An

increase of BDw indicates that the foam structure is less homogeneous.

I-4 Results and discussion

I-4.1 CMC determination and fitting of interfacial tension isotherms using the mod-

ified Frumkin model

QS, GYP, ESC and GA showed a low CMC ranging from 0.008 to 0.015 wt%, see Table

I-5. In contrast, high CMC values were analyzed for TS and TT with 0.5 %wt and 0.1 %wt,

respectively. QS is well known for its low CMC, previous studies reported varying CMC

values of 0.025 wt% (Stanimirova et al. 2011), 0.013 and 0.198 g/L (Wojciechowski 2013)

and 0.5-0.7 g/L (Mitra and Dungan 1997). The differences were explained by the purity

and variation in the saponin composition. There is no literature on the surface activity of

Gypsophila saponins available, but studies on the molecular structure show that QS and

GYP consist of a similar aglycone structure (see Table I-1). The CMC for ESC is in good

agreement with Golemanov et al. (2013), who used an extract from the same supplier, bo-

tanical origin and purity. In contrast, Golemanov et al. (2013) reported lower CMC values

for TS and TT of 0.017 wt% and 0.048 wt%, respectively. The difference can be explained

by the botanical origin. Although extracts were obtained from the same plant, Golemanov

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et al. (2013) used extracts from other parts of the plants, which may result in a different

saponin composition and therefore different CMC values.

Table I-2 Fitting parameters of the experimental data using the Frumkin model

Average MW*

[10

m²/mol]

[nm²]

target

[m³/mol]

QS 1850 2.6 1.5 0.43 0.23 7.8E+02

GYP 1550 4.7 2.0 0.77 0.14 5.1E+04

TS 1250 2.8 -1.2 0.47 0.37 3.2E+04

ESC 1100 3.5 0.3 0.58 0.17 4.6E+04

GA 839 3.5 1.5 0.58 0.10 2.5E+03

TT 850 4.0 -2.1 0.66 0.40 3.5E+04

* based on literature review in Table I-1

In Table I-2 the fitting parameter of the interfacial adsorption isotherms for the six saponins

are displayed. The calculated molecular area A, derived from ω0, of the different saponins

at the interface ranged from 0.43 to 0.77 nm² for QS and GYP, respectively. Although QS

consists of saponins with the highest molecular weight, the packaging is more dense com-

pared to all other saponins. For QS the determined area per molecule A is in agreement with

previous studies, which reported 0.30 nm² and 1 nm² for other QS extracts (Stanimirova et

al. 2011; Wojciechowski et al. 2011). The parameter ε was for saponin extracts below

0.005 m/mN, thus the surface coverage θ of the saponin interfaces does not depend on the

surface pressure Π. For the interaction parameter a the saponins TS and TT have a negative

value and QS, GYP and GA a positive value. In the literature there are different interpreta-

tions of the interaction parameter a. Some authors interpret a positive value as a sign for

attraction between the molecules and a negative value as a sign for repulsion (Kolev et al.

2002; Karakashev et al. 2004). However, Fainerman et al. (1998) clarified that the interac-

tion parameter is rather a modeling parameter and cannot be interpreted in that way.

I-4.2 Short-term adsorption using two-fluid needle experiments

Interfacial tension as determined by the Wilhelmy plate characterizes the interface in a

quasi-static state. Kinetics of interfacial adsorption can be determined by dynamic

interfacial tension measurement. However, in the classical approach in a pendant drop

tensiometer, the interface is already partly covered by adsorbed molecules at the time a

sufficiently high droplet volume is generated to start the calculation (Rodriguez et al. 2005).

Tamm et al. (2012) recently described a two-fluid needle system to investigate the

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adsorption behaviour of proteins at the interface, in which a protein solution is injected into

an existing droplet of water. Thus, the two-fluid needle experiments give more insight on

the short-term adsorption characteristics, which may be correlated to the foaming process.

The lag-time and the diffusion coefficient can be derived from the development of the

surface pressure after the injection of saponins (see Figure I-1 and Table I-3). TT, GA and

TS occupied the interface very fast with lag-times of 0.6 s, 1.7 s and 2.9 s, respectively. In

contrast, QS had a long lag-time of 12.4 s, which is at least two times higher compared to

all other saponins. The differences in the varying lag-time may be explained by the different

molecular weights of the saponins. Although no detailed information on the composition

of the extracts were available in the present study, general data on the molecular weight can

be derived from the literature (see Table I-1). QS and GYP contain saponins with high

molecular weight of 1300-2300 g/mol. TS and ESC contain saponins with an intermediate

molecular weight (~1200 g/mol) and GA contains a saponin with a molecular weight of

839 g/mol. TT extract has a high and low molecular fraction with 1100 g/mol and 600

g/mol, respectively. Saponins with a high molecular weight may need more time to reach

the interface in comparison to saponins with low molecular weight.

Table I-3 Short- and midterm adsorption parameters of six saponin extracts at twofold CMC pH 5

Lag time

[s]

[mN/m]

1800s

[mN/m]

/Π

1800s

[%]

[nm]

calc

[m²/s]

Dexp [m²/s]

12.4 ± 1.5

15.4 ± 0.7

38.2 ± 0.7

47.8 ± 2.1

1.5

1.41E-10

2.5E-11 ± 0.9E-11

GYP

6.1 ± 0.5

15.0 ± 0.9

40.4 ± 0.7

50.9 ± 3.8

1.3

1.61E-10

5.8E-11 ± 1.5E-11

2.9 ± 1.5

23.1 ± 0.8

37.0 ± 1.0

67.8 ± 2.0

1.9

1.11E-10

1.1E-11 ± 0.2E-11

ESC

6.0 ± 0.8

18.4 ± 0.3

42.0 ± 1.4

60.2 ± 1.9

1.7

1.23E-10

3.4E-11 ± 1.3E-11

1.7 ± 0.8

9.6 ± 1.2

61.3 ± 0.3

69.1 ± 7.7

1.3

1.59E-10

1.0E-12 ± 0.3E-12

0.6 ± 0.2

15.7 ± 0.5

51.8 ± 1.9

76.7 ± 2.3

2.0

1.08E-10

5.4E-15 ± 2.0E-15

In addition, it is interesting to compare the surface pressure 5 s after the start of its increase

in relation to the surface pressure after 1800 s (Π5s/Π1800s, Table I-3). At a high ratio, the

equilibrium interfacial tension is reached very fast. In contrast, a low ratio indicates that

rearrangements at the interface slow down the incorporation of additional surfactant mole-

cules and more time is required to reach equilibrium interfacial tension. 5 s after the start

of the increase of the surface pressure the monodesmosidic saponins TS, ESC, TT and GA

reached at minimum 60 % of the equilibrium value. In contrast, the bidesmosidic saponins

QS and GYP reached less than 50 % of the equilibrium value, indicating that it takes longer

for the bidesmosidic saponins to arrange at the interface because the additional sugar resi-

due negatively affects the kinetics of the adsorption at the interface.

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Figure I-1 Surface pressure Π versus square root of the drop age t1/2 at the air/water interface of

QS (◆), GYP (▲), TS (×), ESC (●), GA (■) and TT (+) from different botanical ori-

gins at pH 5, twofold CMC

A theoretical value for the diffusion coefficient from the subsurface to the surface of all six

saponins was calculated based on the estimated maximum length of the molecules. Table

I-3 shows that the roughly estimated radii of the different spherical shapes ranged from 1.3

to 2.0 nm resulting in calculated diffusion coefficients Dcalc between 1.11E-11 m²/s and

1.61E-11 m²/s. These coefficients imply a rather similar adsorption behavior for the saponin

extracts. All experimentally derived diffusion coefficients were lower than the calculated

values indicating that the diffusion at a short-term scale is not only diffusion-limited but an

additional adsorption barrier exists. The difference between Dcalc and Dexp was smallest for

GYP and highest for GA and TT. The presence of an additional adsorption-barrier was re-

ported for QS extracts before (Wojciechowski et al. 2011).

-5

050 100 150

Π[mN/m]

t 1/2 [s1/2]

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I-4.3 Foaming, foam stability and foam structure

As expected, with respect to formation of the foams the saponins with a high diffusion

coefficient (QS, GYP, TS and ESC) had a high foaming speed kf and immobilized larger

amounts of liquid indicated by fden,f-max (see Table I-4). TT was not foamable at twofold

CMC and was not considered in all further experiments. GYP had the highest foaming

speed with 1.81 mm/s, see Figure I-2A, but in general the foaming speed was in a similar

range for all samples. However, the resulting foam structure was very different for GA

compared with all other foams (Figure I-2C). In Figure I-2B, it can be seen that the saponins

TS, GYP, QS and ESC incorporated 10 to 12 % of liquid. GA only incorporated 6.8 % of

liquid. The difference in foam structure was also reflected in the data on transmissibility

(BDm,80s), see Table I-4. QS, GYP, TS and ESC all had similar values for transmissibility

and a low value for BDw,80s at the end of foaming, which indicates a homogeneous and

dense bubble distribution.

Figure I-2 Foam characterization with the parameters a) foaming speed (kf) and b) foam density

at f-max (fDen,f-max) and (c) foaming pictures after 80 and 1800 s of foaming of QS,

GYP, TS, ESC and GA at twofold CMC

QS GYP TS ESC GA

kf[mm/s]

QS GYP TS ESC GA

fDen,f-max[%]

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In terms of foam stability, foams made from QS, GYP, TS and ESC were more stable and

dense than foams made from GA. Foams made from QS, GYP and TS were most stable

and after 3600 s over 85 % of the initial foaming height was still present. Foam stability of

an extract of Camellia oleifera (here TS) was compared to the conventional foaming agents

Tween 80 and SLS by Chen et al. (2011). Foams made from ESC were also stable, but only

77.2 % of the initial foam height was present after 3600 s, see Table I-4. A possible expla-

nation is the more heterogenic bubble distribution of ESC foams at the beginning of the

experiment compared to QS, GYP and TS based foams, see Figure I-2C.

In ESC-based foams, therefore in addition to coalescence, disproportionation occurred

over time, where large bubbles grow and small bubbles disappear by diffusion of dissolved

gas through the foam films. The comparison of foam density values does not show any

differences though the foams vary tremendously, see Table I-4. The differences in homo-

geneity of the foam were visible when comparing the values for BDw throughout the

measurement, see Table I-4. The BDw values of foams made from ESC increased from 20.0

at 80 s up to 124.5 at 3600 s. In comparison: the BDw values of foams made from GYP only

increased from 20.5 at 80 s to 34 at 3600 s. These factors account for the higher foam decay

observed for ESC. The difference in homogeneity can only be quantified in the current

experimental setup, when analyzing the brightness distribution.

Table I-4 Results from foam experiments of QS, GYP, TS, ESC and GA at twofold CMC, pH 5

Parameter QS GYP TS ESC GA

Foam

stability

f1800s [%] 94.2 ± 0.4

93.1 ± 0.8

92.8 ±0.1 89.9 ± 0.5 0.0 ± 0.0

f3600s [%] 93.9 ± 0.2

92.6 ± 0.7 86.4 ±0.1 77.2 ± 2.5 0.0 ± 0.0

Foam

structure

BDm,80s [-] 62.5 ± 0.7

60.0 ± 0.0

61.5 ± 0.7 59.0 ± 0.0 58.0 ± 0.0

BDm,1800s [-] 70.5 ± 2.1

68.5 ± 0.7 66.5 ± 0.7 77.0 ± 2.8 136.0 ± 53

BDm,3600s [-] 73.0 ± 4.2

71.0 ± 1.4 68.5 ± 0.7 99.0 ± 2.8 0.0 ± 0.0

BDw,80s [-] 20.5 ±0.7

20.5 ± 0.7 21.0 ± 0.0 20.0 ± 0.0 20.0 ± 1.4

BDw,1800s [-] 30.5 ± 0.7

28.5 ± 0.7 26.5 ± 0.7 37.5 ± 9.2 151.0 ± 1.4

BDw,3600s [-] 35.5 ± 3.5

34.0 ± 0.0 32.5 ± 0.7 124.5 ± 16 0.0 ± 0.0

fden,f-max [%] 12.3 ± 0.0

12.3 ± 0.1 12.9 ± 0.7 10.5 ± 0.4 6.8 ± 0.1

fden,1800s [%] 6.4 ± 0.1

5.7 ± 0.1 6.0 ± 0 7 5.9 ± 0.5 43.8 ± 5.0

fden,3600s [%] 6.3 ± 0.2

5.6 ± 0.2 6.2 ± 0.7 6.5 ± 0.3 0.0 ± 0.0

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Foams made from GA fully collapsed in less than 20 min. The low foam stability of foams

made from GA can be explained by the low ability of GA to reduce the interfacial tension,

the small amount of incorporated liquid inside the foam and the poor solubility due to the

small hydrophilic part in the molecule.

It is tempting to draw conclusions from the two-fluid needle-experiments on the short

term adsorption and the foaming experiments of the different saponin extracts. In both ex-

periments an empty surface is occupied by a surfactant. The two-fluid needle experiment

is a rather static experiment. In contrast, foaming is a dynamic process with foam develop-

ment and simultaneous foam decay. In addition, wall adhesion and gravitational forces

affect the results of the foaming experiments. These differences have to be kept in mind

when comparing the two-fluid needle experiments with the foaming experiments. Tamm et

al. (2012) found a high correlation between the slope of the Π/t0.5-graph and the foam

weight for different degrees of hydrolysis and concentrations of whey proteins, but for

complex mixtures like the saponin extracts in the present study no clear correlation was

found. However, the calculated diffusion coefficient derived from the two-fluid needle ex-

periments, were in line with the foaming results.

I-4.4 Influence of pH and salt on interfacial tension and foam properties

After basic characterization of the interfacial tension behavior and foam properties of the

six saponin extracts, it is important to analyze the behavior under different environmental

conditions like pH reduction and addition of NaCl. One important question we had to an-

swer in this context is, whether the saponins with a limited number of carboxylic groups

behave like an ionic or non-ionic surfactant. The main advantage of non-ionic surfactants

is their insensitivity to changes in ionic strength, because there are no dissociable groups

inside the molecule. In the past there have been several approaches to determine whether

saponin extracts behave more like ionic or non-ionic surfactants, but results have been con-

tradictory (Wojciechowski 2013).

In the present study for the saponins QS, GYP, ESC and GA a breaking point was de-

tected and therefore these extracts were classified as ionic surfactants, see Table I-5. The

derived CMC values (CMCcond) ranged between 0.011 and 0.023 %wt and were higher than

the determined CMC values from interfacial tension measurements. For TS and TT there

was no breaking point detectable.

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Table I-5 CMCcond, CMCSFT, slope below (kbelow), slope above (kabove) the CMC and ratio of both

(kbelow/kabove) for QS, GYP, ESC and GA; for TS and TT no breaking point was detected

CMCSFT [%wt] CMCcond [%,w/v] kbelow kabove kbelow/kabove

QS 0.008 0.012 2537 1905 1.3

GYP 0.014 0.011 2900 2582 1.1

TS 0.050 / 527 /

ESC 0.009 0.022 151 69 2.2

GA 0.015 0.023 2123 735 2.9

TT 0.106 / 2534 /

SDS 0.27* 0.24 1780 898 2.0

*%w/v (Khan and Shah 2008)

TS is described in the literature to behave like a non-ionic surfactant, although the literature

on molecular structure reported several molecules in TS extracts with a carboxylic group

(Feng et al. 2015), see references in Table 1. The literature on the structure of TT did not

report a COOH-group in the different molecules. To confirm the absence of carboxylic

groups in the extracts, FTIR spectra were recorded and analyzed concerning C═O-bonds.

For both extracts no peak in the C═O binding region in the FTIR spectra was detectable,

see Figure I-3.

Figure I-3 FTIR spectra of C═O binding region (1718-1731 cm-1) of QS (◆), GYP (■), TS (▲),

ESC (●), GA (+) and TT (x)

0,25

0,5

0,75

16001650170017501800

Transmission

Wavenumber [cm-1]

0.75

0.25

0.5

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The change in pH from pH 5 to 3 and the addition of NaCl decreased the interfacial tension

of solutions made from QS, ESC and GA at 0.5-fold CMC, see Figure I-3. The interfacial

tension of solutions made from GYP, TS and TT were less affected by the different envi-

ronmental conditions. GYP was prior classified as an ionic surfactant for which the

interfacial tension should be affected by changes in pH and ionic strength. But GYP had a

much lower peak in the C═O region of about 1730 cm-1 in the FTIR spectra than QS, ESC

and GA, which indicated a lower number of carbonyl or carboxylic groups inside the ex-

tract. FTIR-spectra with distinct peaks at around 1730 cm-1 were reported for different QS

extracts by Almutairi and Ali (2015). It can be speculated that only a small amount of car-

boxylic groups is present in GYP and the peak in the C═O region is mainly due to carbonyl

groups, see Figure I-3.

Figure I-4 Influence of pH and ionic strength on interfacial tension σ at 0.5-fold CMC

The influence of a change in pH and ionic strength were different when comparing foam

results (see Figure I-4). To evaluate foam destabilization and foam stabilization all experi-

ments were performed at a saponin concentration of 0.5-fold CMC. A low concentration of

saponin was chosen to determine not only negative effects like fast foam destruction but

also positive effects like foam stabilization. At higher concentrations, like twofold CMC,

some foams were extremely stable and a positive effect would not be detectable. The values

at pH 5 for foam decay after 3600 s (f3600s), foaming speed (kf) and the parameters describ-

ing foam structure (BDm,600s; BDw,600s and fden-fmax) were taken as reference value and set as

1. The values of the foam parameters at pH 3, 100 mM and 500 mM were calculated in

QS GYP TS ESC GA TT

σ[mN/m]

pH 5

pH 3

100 mmol

500 mmol

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relation to the values obtained at pH 5, see Figure I-5. Foams made of QS were more stable

at pH 3 and increased ionic strength but the foams were less dense and more heterogenic

in structure. Foams made from GYP were less stable, less dense and more heterogenic un-

der the same conditions. But the influence of the environmental conditions on the foam

properties of QS and GYP were very small compared to foams made from TS, ESC and

GA.

Figure I-5 Influence of decreased pH and increased ionic strength by the addition of NaCl on

foaming speed (kf), foam decay after 3600s (f3600s) and foam structure (BDm,600s,

BDw,600s and fden-fmax) of A) QS, B) GYP, C) TS and D) ESC at 0.5-fold CMC; pH 5 -

black solid line (reference values set as 1), pH 3 – gray solid line, 100 mM – gray dot-

ted line, 500 mM – gray dashed line

The reduction of pH 5 to pH 3 significantly stabilized the foams made of GA, see Figure I-

6 upper panel. At pH 3 the majority of the carboxylic groups are non-dissociated and the

electrostatic repulsion is significantly reduced. The effect of ionic strength on the stability

of foams is rather complex and yet not fully understood. It is known, that the addition of

neutral ions shields the charges inside the molecule, which reduces electrostatic repulsion

3600s

m,600s

w,600s

den-fmax

3600s

m,600s

w,600s

den-fmax

3600s

m,600s

w,600s

den-fmax

3600s

m,600s

w,600s

den-fmax

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(Pavan et al. 1999). This could explain the positive influence of high ionic strength on GA

(results not shown). The three carboxylic groups are negatively charged at pH 5 and the

addition of ions can shield these groups and foam stability is increased. But this effect is

only small compared to the impact of pH reduction on foams made from GA.

Figure I-6 Foam pictures of GA at 0.5-fold CMC after 80s of start of foaming at pH 5 (upper left)

and pH 3 (upper right) and TS at 0.5-fold after 600 s of start of foaming at 0 mM (lower

left) and 500 mM (lower right)

The presence of NaCl lead to a full decay of foams made from ESC and TS in less than

600 s, see Figure I-6 lower panel. The negative influence of the presence of electrolytes on

saponin foams was reported before (do Canto et al. 2010). When comparing interfacial

tension values, for the non-ionic TS there was no change when increasing ionic strength.

In contrast, the increase of ionic strength lowered the interfacial tension of the ionic ESC.

But foams made from TS and ESC were both tremendously influenced by changes in envi-

ronmental conditions. However, the foams of the ionic saponins QS and GYP were less

affected by increasing ionic strength. It can be speculated that the bidesmosidic saponins

QS and GYP have an optimized packaging at the interface because of the additional sugar

residue, compared to the monodesmosidic saponins TS and ESC, which prevents the foams

from negative effects of environmental changes. In summary the results show, that the con-

cept of classifying saponins in non-ionic and ionic surfactants is not practical in order to

forecast foam properties in the presence of ionic molecules.

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I-5 Conclusions

We showed that the botanical origin of six different saponins had a high impact on interfa-

cial and foam properties. With respect to the methodology, the variables lag-time, Π5s/Π1800s

and CMC, as well as the area per molecule and the interaction parameter from the Frumkin

model had only limited ability to predict foaming properties. Foaming properties of sapo-

nins were more correlated to the experimental diffusion coefficient and its relation to the

calculated values showed that adsorption was controlled by an additional adsorption bar-

rier. However, other factors like simultaneous foam decay have to be taken into account

and may limit the use of the experimental diffusion coefficient in more complex systems.

Furthermore the concept of classifying saponins in ionic and non-ionic surfactants as the

basis for characterization of foam properties seems to be questionable, since saponins clas-

sified as ionic surfactants are not necessarily sensitive to changes in ionic strength.

This study is another step to increase our understanding of the impact of botanical origin,

and thus molecular structure and composition of saponins on the interfacial behavior. Com-

prehensive papers on this relationship are rare and future experiments should focus on

investigating more botanical sources to perform statistically meaningful correlations be-

tween molecular structure and interfacial properties. For this purpose preparative

techniques should be used to fractionate saponin extracts and with HPLC measurement the

quantitative composition of saponin extracts should be analyzed. The analytical results can

be correlated to interfacial tension measurements, oscillation results and short-term adsorp-

tion. These fundamental results can be used to predict foaming properties. Besides the

analysis of basic interfacial phenomena of saponins, it is also important to increase research

on the application of saponin in foods. Yet, saponins are only approved for some food

products, however, for future applications it is necessary to understand interactions of sap-

onins with other food ingredients like proteins.

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Manuscript II

II Mixtures of saponins and beta-lactoglobulin

differ from classical protein/surfactant-sys-

tems at the air-water interface

Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2016, V. 506,

pp. 765-773. doi: http://dx.doi.org/10.1016/j.colsurfa.2016.07.057.

Authors

Sandra Böttchera

Matteo Scampicchiob

Stephan Druscha

a Technische Universität Berlin, Institute for Food Technology and Food Chemistry

Department of Food Technology and Food Material Science

Königin-Luise-Str.22, 14195 Berlin

b Free University of Bolzano, Faculty of Science and Technology, Piazza Università 1,

39100 Bozen-Bolzano, Italy

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II-1 Abstract

Interactions between surfactants and proteins have been intensively studied in the past be-

cause their interactions in food and cosmetic products can tremendously alter the product

properties. Recent studies have shown that Quillaja saponins (QS) have very different in-

terfacial properties in comparison to common low-molecular weight surfactants. It was

reported that QS forms highly elastic interfacial films, adsorbs more slowly to the interface

and cannot as easily be classified as an ionic or non-ionic surfactant. However, the mecha-

nism of interaction between QS and proteins like beta-lactoglobulin (β-LG) is still to be

understood. For this purpose the present study aimed to explore the interactions between

Quillaja saponin and beta-lactoglobulin in the bulk and at the air/water-interface. At this

purpose, interfacial properties were characterized with dynamic interfacial tension, short-

term adsorption, shear and dilational oscillation experiments and the results were compared

to foam properties. To study molecular interactions fluorescence quenching was analyzed

and sequential two-fluid needle experiments were performed to get more insights on deple-

tion of β-LG by QS.

The presence of β-LG lowered the dilational viscoelasticity of the mixed films. Although

the interfacial film was weakened by β-LG the dilational elasticity was still very high and

did not result in reduced foam stability. Interfacial shear results suggest that QS and β-LG

can interact at the interface probably through hydrogen bonds and/or hydrophobic interac-

tions. We determined 1.7 binding sites on β-LG in the bulk which are accessible for

complex formation for QS by fluorescence quenching experiments. But these interactions

did not affect interfacial properties at the analyzed concentrations. Results of the sequential

two-fluid needle experiments indicated interactions between β-LG and QS at the interface.

The findings of this work can contribute to a better understanding of the properties of com-

plex systems with mixtures of natural surfactants and proteins.

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II-2 Introduction

Quillaja saponins (QS) are surface active natural foaming agents, which consist of a hydro-

phobic triterpenoid agylcone with hydrophilic esterified sugar moeties. QS has a molecular

weight of around 2000 Da and is smaller than most proteins but larger than low-molecular

weight surfactants (<500 Da). The molecular structure of QS is more complex than the

intensively studied common surfactants, like anionic SDS (Fainerman et al. 2010), cationic

CTAB (Phan et al. 2012) or non-ionic CnDMPO (Ivanov et al. 2010). Saponin extracts

consist of a variety of derivates, which differ in amount, position and type of sugar residues

as well as the type of aglycone. Differences in saponin composition even between different

QS extracts affect interfacial properties tremendously (Golemanov et al. 2013;

Wojciechowski 2013). Although QS possess chargeable carboxylic groups, the amount is

very small in relation to the molecular size of the QS molecule. The classification of QS as

an ionic or non-ionic surfactant is therefore, not as straightforward as for other surface

active molecules. But, as we reported before (Böttcher and Drusch 2016) Quillaja saponins

behave more like ionic surfactants as determined by conductivity measurements. However,

it has to be kept in mind that not all assumptions or explanations for ionic surfactants may

be valid for QS because of the more complex and differing molecular structure. The high

dilational (Stanimirova et al. 2011) and shear moduli (Golemanov et al. 2012) distinctively

discriminate QS from common surfactants, which have rather poor dilational and shear

viscoelastic properties (Mackie et al. 2000; Fainerman et al. 2010). Quillaja saponins are

excellent foaming agents due to their high surface activity and QS foams exhibit a high

stability over a long time because of the formation of viscoelastic interfacial films (Böttcher

and Drusch 2016).

Beta-lactoglobulin (β-LG) is a well-studied surface active globular whey protein with a

hydrophobic calyx in which small hydrophobic molecules can enter. At pH 7 (pH>isoelec-

tric point), β-LG exists as a dimer with an overall negative charge (Creamer et al. 2011).

Proteins in general and β-LG in particular can lower the interfacial tension, but adsorption

is slower compared to common low-molecular weight surfactants (Wierenga and Gruppen

2010). β-LG can sufficiently stabilize foam lamellas by forming highly viscoelastic inter-

facial films (Petkov et al. 2000).

Interactions between low-molecular weight surfactants and proteins have been exten-

sively studies in the past (Bos and van Vliet 2001). Complex formation in the bulk is

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analyzed because complexation can influence interfacial properties like for example be-

tween diacteyltartaric esters of monoglycerides (DATEM) and whey proteins in bread

dough. In mixtures, high foam stability of proteins is reduced by the addition of low-mo-

lecular weight surfactants. Experiments on the interaction between β-LG and the anionic

surfactant SDS showed that SDS reduces the film viscoelasticity and leads to reduced foam

stability (Pradines et al. 2009; Lech et al. 2014). The reduction in foam stability can be

explained by the conflictive mechanisms of foam stabilization. Low-molecular weight sur-

factants stabilize foam lamellas by the Gibbs-Marangoni-mechanism (Wilde et al. 2004).

When concentration gradients occur at the air/water-interface, surfactants will instantly

move along the interface or diffuse from the bulk to areas of lower concentration. Simulta-

neously continuous phase is dragged along by the rapid movement of the surfactants. For

this mechanism, the surfactant must have a high lateral mobility, which excludes the pos-

sibility of interactions between neighboring surfactant molecules at the interface. The

surfactants disturb the linked protein network, which reduces essential intermolecular in-

teractions between the proteins. Simultaneously, surfactants cannot level out interfacial

tension gradients because the immobile protein molecules reduce the surfactants lateral

mobility (Maldonado-Valderrama and Patino 2010).

The properties of the mixture of QS and β-LG differ from those of common surfactant/β-

LG-mixtures because of the unique interfacial properties of QS. In contrast to common

surfactants QS molecules form a high viscoelastic network at the interface through interac-

tions between the sugar moeties of neighboring QS molecules (Stanimirova et al. 2011). It

is assumed that the sugar moeties of the QS and the side chains of the β-LG can interact as

well through hydrogen bonds and form a high viscoelastic network at the interface. We

therefore, hypothesize a synergistic effect in foam stability in β-LG/QS-mixtures because

foam stabilization mechanisms of β-LG and QS are similar and not conflictive like in com-

mon surfactant and β-LG-systems. Until now foam stability of mixed β-LG/QS foams was

not reported, but Kezwon and Wojciechowski (2014) showed that no synergistic effect in

foaming can be detected. The authors also proposed, according to their interfacial tension

results, that molecular complexes are formed between QS and β-LG. To the best of our

knowledge no studies determined the interfacial rheology of mixed β-LG and QS-layers

and linked these properties to foam experiments.

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The main aim of this study is to investigate whether QS and β-LG can form a viscoelastic

interfacial network, which results in increased foam stability as well as high shear and di-

lational moduli. Therefore, QS concentration was fixed at 0.005 % (below the CMC) and

increasing concentrations of β-LG ranging from 0.005 to 0.1 % were added. General effects

of the presence of β-LG in QS solutions on the dynamic interfacial tension, short-term ad-

sorption, dilational viscoelasticity were evaluated using drop shape analysis. Shear

viscoelasticity was analyzed using a rheometer equipped with a bicone tool. In addition,

we wanted to find more evidences on whether and how saponins and β-LG interact and can

form complexes. To better understand the interactions of QS and β-LG at the interface two-

fluid needle experiments were performed. Molecular interactions in the bulk were further

analyzed by fluorescence measurements.

II-3 Material and methods

An extract from the bark of Quillaja saponaria Molina (QS) was provided by Ingredion

Germany GmbH (Hamburg, Germany). The QS extract in this study has been analyzed

sufficiently considering interfacial properties and chemical structure (Tippel et al. 2016a).

The extract was further purified by solid phase extraction (SPE) with a C-18 column

(10 g/70 mL Thermo Fisher Scientific Germany BV & Co KG; Braunschweig, Germany)

to a purity of 81 %. β-Lactoglobulin (β-LG) was isolated from whey protein isolate (Fon-

terra DSE 6668). Therefore, α-lactalbumin, bovine serum albumin and immunoglobulin

were precipitated at pH 3.8 and the supernatant was freeze dried. The extracted β-LG had

a purity of 89 %. For interfacial tension measurements, short-term adsorption, dilational

and shear rheology as well as foam experiments a QS concentration of 0.005 % and in-

creasing concentrations of β-LG: 0 (only QS), 0.005 (1:1), 0.01 (2:1), 0.05 (10:1) and 0.1 %

(20:1) were analyzed. All solutions were prepared using 10 mM phosphate buffer (pH 7)

and made at least 8 h before measurement and continuously stirred.

II-3.1 Purification of saponin extract

The saponin extract was purified and fractionated by a modified method using solid phase

extraction as described before by Reim and Rohn (2015). At first methanol insoluble con-

tents were separated to prevent sedimentation during solid phase extraction. Therefore, the

extract was diluted with a sufficient amount of methanol and afterwards centrifuged at

5000 g. The clear supernatant was evaporated and diluted with distilled water to a saponin

concentration of about 2 %. The column was conditioned with 70 mL of pure methanol and

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equilibrated with 70 mL distilled water. 70 mL of diluted saponin solution was rinsed

through the column and afterwards the column was washed with 35 mL of a mixture of

water:methanol (95:5) and sucked dry with vacuum. Five elution steps were performed

using 35 ml each of 40, 50, 60, 70 and 100 % alkali methanol. Fraction II-V were pooled,

methanol was evaporated and samples were freeze-dried.

II-3.2 Fluorescence quenching of β-LG by the presence of QS

The fluorescence of proteins, more precisely the fluorescence of the tryptophan residues,

can be altered by the presence of a quencher. Either static or dynamic quenching can occur.

Dynamic or collisional quenching describes the interactions of protein and quencher only

during the lifetime of the exited state. Static quenching occurs in the ground-state (non-

exited stated) when proteins and quencher form complexes, which are non-fluorescent. In

this study QS is the quencher, which alters the fluorescence of β-LG. The main contributor

to the emission intensity of native beta-lactoglobulin is Trp-19, which is situated inside the

calyx in a rather hydrophobic environment. The second tryptophan residue Trp-61 is lo-

cated at the binding site of the dimer and only minor contributes to the overall emission.

(Viseu et al. 2007)

To distinguish between static and dynamic quenching the absorption spectra between

250-300 nm of QS, β-LG and their mixtures were analyzed (Shpigelman et al. 2010). If the

adsorption spectrum of β-LG is not altered in the presence of QS then it is highly likely that

dynamic quenching occurs, but if the adsorption of β-LG is reduced in the presence of QS

it is highly likely that static quenching occurs and ground-state complexes are formed.

To determine the quenching of β-LG by QS fluorescence measurements were performed

using a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies Deutschland

GmbH &Co. KG, Waldbronn Germany). The samples were excited at 295 nm (wavelength

to excite tryptophan residues) and emission was recorded at 336 nm. 10 mM PBS buffer

was used as blank. The self-fluorescence of QS and dilution of β-LG were determined by

blank experiments. All experiments were conducted at 20°C, with excitation and emission

slits of 5 nm and exited at 650 V. Samples were checked for inner filter effects and cor-

rected according to the Eq. (I-1) stated by van der Weert (2010)

𝐹𝐹𝑐𝑐𝑐𝑐𝑟𝑟𝑟𝑟 =𝐹𝐹𝑐𝑐𝑜𝑜𝑜𝑜 ∙10𝐴𝐴𝑒𝑒𝑒𝑒𝑒𝑒+𝐴𝐴𝑒𝑒𝑒𝑒

2 (I-1)

where Fcorr is the recalculated fluorescence value, Fobs is the measured fluorescence value

and Aexc (here: 295 nm) and Aem (here: 336 nm) are the absorption values at the excitation

and emission wavelength, respectively.

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The adsorption values were determined using Helios Omega UV-VIS Spectrophotometer

by Thermo Fisher Scientific Germany BV & Co KG (Braunschweig, Germany). Maximum

binding sites were determined using the Cogan-plot. The quenching data can be analyzed

using Eq. (I-2) and (I-3).

𝑃𝑃∙𝛼𝛼=1∙𝐿𝐿𝑡𝑡𝛼𝛼

𝑛𝑛∙(1−𝛼𝛼)−𝐾𝐾′𝑑𝑑

𝑛𝑛 (I-2)

𝛼𝛼=𝐹𝐹𝑒𝑒𝑚𝑚𝑒𝑒−𝐹𝐹𝑒𝑒𝑐𝑐𝑐𝑐𝑐𝑐

𝐹𝐹𝑒𝑒𝑚𝑚𝑒𝑒−𝐹𝐹0 (I-3)

where P is the β-LG concentration, Lt is the QS concentration, K’d is the apparent dissoci-

ation constant, n the maximum number of binding sites, Fmax and F0 are the fluorescence

intensities upon saturation and initial β-LG-concentration, respectively. Therefore, a plot

with Lt∙α/(1-α) the x-axis and P∙α on the y-axis is created. The quantity of maximum bind-

ing sites is 1/slope.

II-3.3 Dynamic interfacial tension measurements, dilational rheology, short-term ad-

sorption and sequential two-fluid needle experiments

For dynamic interfacial tension measurements the pendant drop mode of drop shape anal-

ysis system OCA-20 from DataPhysics Instruments GmbH (Filderstadt, Germany) was

used. A droplet with a volume of 15 µL was automatically dosed at the tip of a needle

(d=1.65 mm) and the dynamic interfacial tension was recorded for 30 min. The system

calculates the interfacial tension by fitting the Young-LaPlace-equation to the drop shape.

Sinusoidal volume drop expansion and compression experiments were performed using the

oscillation unit ODG-20 to analyze dilational rheology. A 15 µL drop was created at the tip

of a needle and first oscillation cycle was started 30 s after drop creation. In each cycle the

drop was oscillated six times at a frequency of 0.1 Hz with 2.8 % of volume amplitude,

which resulted in an area change ΔA/A. After a waiting time of 30 cycles (300 s) sequential

oscillation was performed up to 20 times (Wan et al. 2014). From the data of each cycle

the interfacial dilational modulus E* was calculated with E’ and E’’ representing the elastic

(storage) and viscous (loss) modulus, respectively.

To study short-term adsorption two-fluid needle experiments were performed (Tamm et

al. 2012). For these experiments a needle with a diameter of 0.51 mm was placed inside a

needle with a diameter of 1.65 mm. A droplet of 14 µL of distilled water was manually

dosed and an aliquot of 1 µL of the surfactant solution was automatically dosed into the

existing water droplet. Adsorption was monitored for 10 s at 130 fps with a CCD camera.

QS and β-LG-concentrations were adjusted to achieve standard concentrations after injec-

tions into the droplet. Transport through the droplet was characterized by the lag-time,

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which is the time between start of volume expansion (starting volume+1 % volume expan-

sion) and the drop of the interfacial tension (surface pressure>0.5 mN/m) and was discussed

in several publications before (Tamm et al. 2012; Böttcher and Drusch 2016). The diffusion

from the subsurface to the interface can be determined by plotting the surface pressure over

square root of the time (t1/2).

Sequential two-fluid needle experiments were performed by manually dosing a 14 µL

droplet of a 1 % β-LG-solution through the large needle of the two-fluid-needle. After 20

min 1µL of a 0.15 % QS-solution was automatically dosed into the existing droplet. After

the injection the interfacial tension was recorded for an additional hour.

II-3.4 Shear rheology

For measurement of interfacial shear rheology the Rheometer Physica MCR301 from An-

ton Paar Germany GmbH (Ostfildern, Germany) was used, which is equipped with an

interfacial rheology system cell (IRS) and a bicone tool. The diameter of the cell was

80 mm and was filled with 120 mL of liquid. The bicone tool had a diameter of 68.25 mm

and an angle of 5.016°. Care was taken to remove all bubble from the solution prior to the

measurement. Film formation was monitored for 9 h with a strain amplitude γ0 of 0.1 %

and frequency f of 1 Hz every 5 min. Samples were measured at 25°C.

Surfactant or protein loaded interfaces exhibit elastic and viscous properties when harmonic

sinusoidal deformation (strain) is applied. The complex shear modulus G*(ω) is defined as

(Krägel et al. 2008): 𝐺𝐺𝑖𝑖∗(𝜔𝜔)=𝐺𝐺𝑖𝑖′(𝜔𝜔)+𝑖𝑖𝐺𝐺𝑖𝑖′′(𝜔𝜔), where ω is the straining frequency and G’

and G’’ are the elastic (storage modulus) and the viscous moduli (loss modulus), respec-

tively.

II-3.5 Foaming, foam stability and foam structure

The foaming, foam decay and foam structure were monitored using DFA 100 (Krüss

GmbH, Hamburg, Germany). The procedure is described elsewhere (Böttcher and Drusch

2016). Briefly, 50 mL saponin or protein solution was foamed using pressurized air flowing

through a porous glass frit with a pore size of 40-100 µm. Foam and liquid height as well

as a brightness profile were recorded twice a second for 3600 s by transmissibility meas-

urement. All experiments were performed under light exclusion and repeated at least twice.

Foam stability was characterized by relative remaining foam height f%,3600 after 3600 s and

foam density was compared after end of foaming fden,fmax.

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II-4 Results

II-4.1 Interactions in the bulk determined by fluorescence quenching

There is evidence that QS and β-LG form complexes in the bulk via static quenching. We

observed a distinctive loss of absorption for β-LG when QS is present (Figure II-1A). As

recently discussed by Keppler et al. (2014) for the quenching of β-LG, there are various

models possible to analyze fluorescence data. The authors recommended to use the Cogan-

Plot and showed that even though the calculated results for the ‘apparent affinity constant’

(K’a) and ‘apparent dissociation constant’ (K’d) showed high variation the ‘maximum

number of binding sites’ (n) was relatively stable. The inverse slope of the graph of the

Cogan-plot is the maximum number of binding sites. We determined a maximum of 1.7

binding sites (Figure II-1B).

Figure II-1 Fluorescence measurements on the interactions of β-LG and QS, A) absorption spec-

tra between 250-300 nm of pure β-LG (solid line), pure QS (dashed line) and mixture

of β-LG+QS, which was corrected for QS adsorption (dotted line), B) Cogan-Plot of

quenching of β-LG by QS with P=β-LG-concentration, α=fraction of free binding

sites and Lt=QS-concentration

250 275 300

Absorption [

Wavelength [nm]

β-LG

β-LG+QS

y = 0.59x -4E-07

R² = 0.94

0,E+00

1,E-05

2,E-05

0,0 0,0 0,0

P*α

Lt*α/(1-α)

-5

2∙10

-5

4∙10-7

2∙10-5

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II-4.2 Interactions of QS and β-LG at the interface

An interfacial layer of β-LG can be penetrated by the addition of QS. In the experimental

setup β-LG can adsorb at the air-water interface for 20 min. When QS is injected into the

droplet, new interface is created (area expansion of about 5 %) for which QS and β-LG

compete (Figure II-2, closed circles). A sharp decrease in interfacial tension after the injec-

tion is visible and reaches almost interfacial tension of pure QS. Afterwards the interfacial

tension increases slightly by about 1 mN/m and after half an hour the interfacial tension

decreases again. At the end of the measurement the interfacial tension value is between

pure QS (Figure II-2, diamonds) and β-LG (Figure II-2, open circles).

Figure II-2 Interfacial tension σ in relation to the drop age t for the injection of 0.15 % QS into a

droplet of 10 mM phosphate buffer (◆) and sequential adsorption of 1 % β-LG at the

air water interface with injection indicated by the arrow of 0.15 % QS (●) and injection

of 10 mM phosphate buffer (○)

01000 2000 3000 4000 5000

σ[mN/m]

t [s]

injection of QS or

buffer

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II-4.3 Short- and midterm adsorption of mixtures of QS and β-LG

The presence of β-LG cannot efficiently in-

fluence short- and midterm adsorption in

mixtures of QS and β-LG. But still QS ad-

sorbs more rapidly than β-LG, as shown in

Figure II-3. The open symbols represent β-

LG and it can be noted that with increasing

concentration of β-LG the adsorption is

faster and surface pressure Π is increasing.

The presence of β-LG neither decreases nor

increases the adsorption of the QS in the

short time scale of 5 s. Lag-time of the dif-

ferent β-LG concentrations reduced with

increasing β-LG concen-tration, but be-

tween mixtures of QS and β-LG and pure

QS no differences were found (results not

shown).

At a timescale of several minutes (midterm adsorption) the dynamic interfacial tension is

also governed by QS (see Table II-1). The interfacial tension of pure β-LG is distinctly

higher than that of QS with 51.2 to 56 mN/m. In mixtures of QS and β-LG the interfacial

tension was lowered similarly to QS to about 45 mN/m.

Table II-1 Dynamic interfacial tension σ of β-LG-solutions and β-LG/QS-mixtures measured for

20 min with pendant drop analysis

cQS [%wt] cBLG [%wt] β-LG/QS [%wt/%wt] σ [mN/m]

0.005 56.0 ± 0.9

0.01 55.0 ± 0.5

0.05 51.7 ± 0.5

0.1 51.2 ± 0.3

0.005

0 45.7 ± 0.8

0.005 1 44.7 ± 1.2

0.01 2 44.8 ± 1.4

0.05 10 45.0 ± 1.2

0.1 20 45.4 ± 0.1

012

Π [mN/m]

1/2

]

increasing β-LG

concentration

Figure II-3 Surface pressure Π versus square root of

the drop age t1/2 at the air/water interface

of 0.005 % QS (+), 0.005 % β-LG (◇),

0.01 % β-LG (△), 0.05 % β-LG (○), 0.1 %

β-LG (□); filled symbols indicate β-LG is

mixed with 0.005 % QS

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II-4.4 Dilational rheology of mixtures of β-LG/QS

Pure QS and β-LG interfacial layers as well as their mixtures build up strong viscoelastic

interfacial layers. In Figure II-4A the complex dilational modulus E* is displayed in rela-

tion to the drop age. Viscoelasticity was highest for QS with about 170-180 mN/m and

lowest for all β-LG-concentrations, which had a viscoelasticity of around 110 mN/m. For

mixtures of QS and β-LG the interfacial dilational modulus E* modulus lies between the

values for QS and β-LG. For all samples the viscous modulus E’’ was below 10 mN/m,

which indicates high elastic properties of the interfacial layers. In mixtures with the highest

concentration of 0.1 % β-LG the complex dilational modulus (E*) is close to the values of

pure β-LG, which indicates that dilational rheology at this concentration is governed by β-

LG.

Figure II-4 Interfacial dilational modulus E* in relation A) the drop age t and B) to the surface

pressure Π of 0.005 % QS (+), 0.005 % β-LG (◇), 0.01 % β-LG (△), 0.05 % β-LG

(○), 0.1 % β-LG (□); filled symbols indicate β-LG is mixed with 0.005 % QS

The development of the complex dilational modulus E* of a surface active substance over

time is not independent from its concentration, therefore E* should be normalized over the

surface pressure Π (Patino et al. 2005). The resulting master curve eliminates concentration

effects on the complex dilational modulus and therefore all β-LG curves should overlap.

Normalization of all four β-LG concentrations was not possible, because E*-Π-graphs were

not similar and no master curve could be created (Figure II-4B). With increasing surface

5.4

5.6 14.5

7.0

9.1

7.1

515 25

Π[mN/m]

100

150

200

02000 4000 6000

E* [mN/m]

t [s]

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pressure, the interactions and therefore E* increased accept for QS whose E* was inde-

pendent from the surface pressure. All protein concentrations had a slope greater than 1,

which increased with increasing β-LG concentration from 5.4 to 7.0 for 0.005 and 0.1 %

β-LG, respectively. In mixtures with the lowest β-LG concentrations 0.005 & 0.01 %, the

slope could not be determined because after a steep ascent the slope became very flat sim-

ilar to QS. Graph characteristics of the two highest β-LG concentrations 0.05 & 0.1 % were

similar to pure β-LG and were without a plateau as described for the mixtures with lower

β-LG concentration.

II-4.5 Shear rheology

Experiments on shear rheology give further insights on intermolecular interactions at the

constant interface. High viscoelastic film properties indicate strong molecular interactions.

We monitored film formation by applying low amplitude and frequency to the interface.

As displayed in Figure II-5, QS builds up strong viscoelastic interfacial layers with

G*~70 mN/m. Interfacial layers of β-LG take more time to develop but with increasing

concentration of β-LG the complex shear modulus G* increases and film formation is

faster. In all samples G’’ was below 10 mN/m and interfacial layers had pronounced elastic

properties. In all QS/β-LG-mixtures, apart from that containing 0.1% B-LG, the complex

shear moduli G* were sufficiently higher than pure QS and pure β-LG, which indicates

additional interactions between QS and β-LG.

Figure II-5 Complex shear modulus G* in relation the interface age t for of 0.005 % QS (+),

0.005 % β-LG (◇), 0.01 % β-LG (△), 0.05 % β-LG (○), 0.1 % β-LG (□); filled sym-

bols indicate β-LG is mixed with 0.005 % QS

100

150

0100 200 300 400 500

G* [mN/m]

t [min]

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II-4.6 Foam properties

Even low β-LG-concentrations can improve properties of QS foams. In mixtures with the

highest β-LG concentration of 0.1 % a foaming speed of 1.745 ± 0.027 mm/s was deter-

mined, which is distinctively higher than the QS foaming speed of 1.671 ± 0.030 mm/s. In

general, QS can form stable and long-lasting foams but in this study a very low concentra-

tion was chosen to obtain unstable foams over the measuring time of 3600 s. Foam stability

of the mixtures was highly influenced by the addition of β-LG. In mixtures containing the

lowest β-LG concentration of 0.005 % the remaining foam height after 3600 s increased to

71.1 % instead of only 21.3 % for pure QS, even though pure 0.005 % β-LG was already

fully collapsed after 3600 s (Figure II-6A). In mixtures the presence of β-LG increased

foam density and thereby the liquid content of the initial foam (Figure II-6B).

Figure II-6 Foam results of QS (red), β-LG (blue) and mixtures of QS and β-LG (violet) for

A) remaining foam height after 3600 s and B) foam density at fmax in relation to β-LG

concentration

00.005 0.01 0.05 0.1

fden,fmax [%]

Concentration β-LG [%]

Mix

B-LG

β-LG

QS+β-LG

100

00.005 0.01 0.05 0.1

3600%

[%]

Concentration β-LG [%]

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II-5 Discussion

At first we want to interpret fluorescence experiments on complex formation in the bulk

and sequential adsorption experiments on the interactions at the interface. In the second

part of the discussion we want to discuss results of foam experiments, adsorption and in-

terfacial rheology.

Fluorescence quenching experiments showed that QS and β-LG can interact in the bulk

via static quenching and a maximum of 1.7 binding sites at the β-LG are accessible for QS

molecules (Figure II-1). Static quenching is most likely related to the formation of non-

fluorescent complexes between QS and β-LG. In the past it was reported that β-LG can

form complexes with naringenin (Gholami and Bordbar 2014) or resveratrol (Liang et al.

2008). Non-covalent binding to the two binding sites in β-LG can occur via hydrogen

bonds, van der Waals forces or hydrophobic interactions (Keppler et al. 2013). The inter-

actions in the bulk were predicted before by interfacial tension data (Piotrowski et al. 2012).

The value of the maximum binding sites is between 1 and 2, which is very common for

reactions of β-LG (Keppler et al. 2013). It also has to be kept in mind that the QS extract

consists of a mixture of various saponins with different molecular structures. A clear stoi-

chiometry is therefore not as straightforward as for a single well defined substance.

Two-fluid needle experiments were performed to determine whether QS can deplete β-

LG from the interface. Fainerman et al. (2005) showed in their two-fluid needle experi-

ments that common surfactants, i.e. C13DMPO and Triton X-100 adsorb and desorb rapidly

from the interface. Proteins, i.e. β-LG take a long time to adsorb and an even longer time

to desorb from the interface, because high activation energy is necessary. But it is possible

to accelerate desorption of proteins by the addition of surfactants (Fainerman et al. 2005).

In the experiment of this study, β-LG was allowed to adsorb at the interface and QS was

added into the droplet. It is clear that because of the addition of QS the interface of the

droplet increases and QS and β-LG compete to adsorb at the unoccupied interface. After

the injection of QS into the β-LG-droplet, a sharp decrease in interfacial tension was ob-

served and, afterwards, the interfacial tension increased by about 1 mN/m reaching a

plateau and decreasing after 30 min again. The results of this study indicate that QS and β-

LG interact at the interface as well. But it is clear that QS cannot fully deplete β-LG from

the interface during the measurement time since interfacial tension of the mixed layer is

higher than pure QS. A possible explanation of these results is that QS and β-LG can either

interact via the ‘complexation and competitive adsorption’ or via ‘orogenic displacement’.

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The ‘orogenic displacement’ describes the adsorption of surfactants molecules at a pro-

tein covered interface (Mackie et al. 1999). First, surfactant molecules adsorb at defects in

the interfacial protein film. Subsequently, additional surfactant molecules adsorb at these

defects and, since proteins do not easily desorb, the protein network is compressed. At a

certain point the protein network cannot be further compressed and protein molecules de-

sorb and the protein network breaks down in favor of a lower interfacial tension obtained

by surfactant molecules. Alternatively, the ‘competitive adsorption and complexation’

mechanism, which was proposed for ionic surfactants: surfactants adsorb at the interface

and form via hydrophobic or electrostatic interactions (or both) complexes with the proteins

(Kotsmar et al. 2009). The complexes are less surface active and are therefore, easier dis-

placed from the interface. Free surfactant molecules and complexes compete for the newly

created space. Over time more and more protein is displaced from the interface through the

complexation and the interface is mainly covered by surfactant molecules.

We cannot verify, which of the two mechanisms takes place but we hypothesize, since

QS behaved more like an ionic surfactant in conductivity measurements, that QS and β-LG

interact by the mechanisms of ‘complexation and competitive adsorption’. After QS ad-

sorbs at the newly created interface, QS molecules interact with β-LG forming less surface

active complexes, which leads to an increase in interfacial tension. After a while the com-

plexes desorb and free space at interface is created and additional QS and β-LG molecules

from the bulk can compete for the newly created interface.

In the second part of the discussion we want to discuss and connect interfacial tension

and rheology results with foam properties.

QS adsorbs faster at the interface than β-LG because of its lower molecular weight as

shown by measurements on short-term adsorption (Figure II-3). This result is in line with

the concept of slow adsorption of protein. Because of electrostatic hindrances and low hy-

drophobicity, it takes time for β-LG to diffuse from the subsurface to the interface

(Wierenga and Gruppen 2010). However, recent studies have shown that QS also has a

lower diffusion coefficient than regular surfactants as well and adsorption is not diffusion-

controlled but an additional adsorption barrier exists (Piotrowski et al. 2012). Adsorption

speeds of mixtures of QS and β-LG do not differ from pure QS showing that QS molecules

clearly dominate the adsorption. Similar results were obtained in dynamic interfacial ten-

sion experiments: the interfacial tension of QS after 20 min was distinctively lower than β-

LG. The interfacial tension of QS/β-LG-mixtures did not differ from QS values (Table II-

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1). In a previous study it was shown that the presence of β-LG can slightly reduce interfacial

tension values (Kezwon and Wojciechowski 2014). We found the same reduction of inter-

facial tension at much lower QS/β-LG-concentrations (results not shown). We attribute the

differences to the use of a different QS extract because Kezwon and Wojciechowski (2014)

also found differences in synergistic behavior between the two analyzed QS extracts in their

study. It seems that the strength of synergistic effect between QS and β-LG depends on the

kind of QS. In addition, Kezwon and Wojciechowski (2014) do not specify whether the

used β-LG is mainly in native state. Partially denaturated proteins can adopt their structure

more freely than native β-LG leading to a smaller area per molecule at the interface (Kim

et al. 2005). In this study only native β-LG was used, which is less flexible and more rigid

in its molecular structure. Therefore, native proteins cannot as easily interact with QS,

forming complexes and optimize interfacial packing, which leads to synergistic effects in

adsorption and interfacial tension.

Interfacial dilational rheology can give insights how an interfacial film can withstand

expansion and compression. Viscoelastic response from the interfacial layer is influenced

by interfacial rearrangements in the interfacial layer and exchange processes between the

bulk and the interface (Miller et al. 1996; Freer et al. 2004).

Interfacial layers of pure QS and pure β-LG were highly viscoelastic E*QS~180 mN/m

and E*BLG~110 mN/m with very low viscous moduli (<10 mN/m) (Figure II-4A). A high

viscoelastic modulus E* with a high elastic modulus of the interfacial layer may be at-

tributed to intermolecular interactions and the inability of desorption from the interface

when subjected to expansion and compression. The low viscous modulus E’’ for all sam-

ples shows that only a small amount of the imposed energy is dissipated by relaxation

processes like structural rearrangements in the interface (Ravera et al. 2009). For different

QS extracts higher and lower values E*QS were reported with 280 mN/m (Stanimirova et

al. 2011) and 80-100 mN/m (Golemanov et al. 2013; Wojciechowski 2013). Petkov et al.

(2000) reported slightly smaller elastic moduli E’ for β-LG-interfaces with E’~66-

90 mN/m. For the sake of comparison typical viscoelasticity for common low-molecular

weight surfactants like SDS are sufficiently lower with E*SDS~10 mN/m as well as the elas-

ticity of random coil proteins like β-casein with E’β-casein~30 mN/m (pH 7) (Fainerman et

al. 2010; Wüstneck et al. 2012).

E* in binary systems of β-LG/QS were lower than pure QS and higher than pure β-LG.

The presence of a small amount of β-LG sufficiently decreased E*, which may be attributed

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to changes in interfacial molecular interactions or differing exchange characteristics be-

tween the interface and the bulk.

The E*-Π-graph shows the dependency of the complex dilational modulus on the surface

pressure (Figure II-4B). A steep slope indicates strong dependence of the E* on the surface

pressure, which means that with increasing surface pressure (because of higher interfacial

surfactant concentration) the viscoelasticity increases. A slope greater than 1 in the E*-Π-

graph indicates non-ideal behavior of the film and high interaction between the molecules

(Lucassen-Reynders et al. 1975). The fact, that it is not possible to achieve a master curve

for β-LG may be attributed to different aggregation behavior at the air/water-interface at

the chosen concentrations. Benjamins (2000) found about the same slope as in our experi-

ments for similar β-LG concentration but in contrast a master curve was reported. This

difference has to be attributed to divergent experimental conditions. The adsorption and

formation of the viscoelastic network of QS is too fast to be determined in the timescale of

our experiments. The same applies for the mixtures with the lowest β-LG concentrations,

which show a fast structuration with an adjacent plateau at higher surface pressures. That

means at low β-LG concentration the dilational interfacial properties are governed by QS.

With increasing β-LG concentration in the mixtures the steepness of the slope decreases

and approaches the steepness of pure β-LG interfacial layers. This indicates that the inter-

facial layers at higher β-LG concentrations are predominantly influenced by β-LG.

With interfacial shear rheology it is possible to determine interaction between molecules

when shear stress is applied to the interface without the influence of relaxation processes

imposed by diffusion to and from the interface (Freer et al. 2004). Similar to dilational

rheology, QS interface was distinctively more viscoelastic (G*QS~70 mN/m) than β-LG-

interfaces (G*BLG~15…75 mN/m), but in both cases elastic moduli G’ were sufficiently

higher than viscous moduli G’’ (Figure II-5). In contrast to dilational rheology, it was pos-

sible to discriminate between the chosen β-LG-concentrations. Complex shear modulus G*

of QS and β-LG-layers in this study were higher than the reported values G*QS~30 mN/m

(Golemanov et al. 2012) and G*BLG~10…20 mN/m (Petkov et al. 2000), which is attributed

to deviating measuring conditions. In mixtures of QS and β-LG (up to a β-LG-concentration

of 0.05 %) the G* was higher than in pure QS and β-LG. At higher β-LG concentrations in

the mixtures the interfacial shear properties are mostly influenced by β-LG and at lower

concentrations by QS. Gunning et al. (2004) determined similar synergistic effects between

the ionic surfactant LPC-L and β-LG. This effect was attributed to the strengthening of the

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protein network by the presence of the surfactant, which increased shear viscoelasticity. It

is assumed that β-LG and QS interact either through hydrogen bonds or hydrophobic inter-

actions or a mixture of both. It is further hypothesized that β-LG can strengthen the firm

QS network at low concentrations but with increasing β-LG concentration this effect is

neglected. But is has to be kept in mind that the viscoelasticity is still very high compared

to common surfactant/protein-systems.

Foam results showed a slightly increased foaming speed and tremendously increased

foam stability and density in mixtures of QS and β-LG compared to pure QS (Figure II-6).

Kezwon and Wojciechowski (2014) focused on analyzing foaming properties and found no

synergistic effect of β-LG on foaming results of QS in the chosen (much lower) concentra-

tion range. Density and stability of the foams in this study were increased by the presence

of β-LG in mixtures with QS. A higher foam density indicates a higher liquid content in

the foam, which can increase foam stability.

Considering the discussed interfacial properties of QS, it is highly likely that QS is not

primary stabilizing foams by the Gibbs-Marangoni mechanism because diffusion is too

slow and lateral mobility not possible, as indicated by high shear and dilational viscoelas-

ticity. We suppose that foam stability of QS foams is attributed to the formed viscoelastic

network. But we also think that in contrast to proteins, QS can stabilize the interfacial films

by diffusion of QS to areas of lower surfactant concentration. Although the QS molecules

cannot laterally move to areas with lower concentration, the diffusion from the bulk to the

interface is faster than β-LG and can additionally stabilize the interface by this mechanism.

In mixtures of β-LG/QS the foam stabilizing mechanisms of both substances are not con-

flictive and therefore, the synergistic effect in foam properties can be explained.

In a last step, we want to compare the results of interfacial shear and dilational rheology

with foam results. In mixtures with a low concentration of β-LG a sufficient increase in

shear viscoelasticity was determined as well as a distinctive decrease in dilational viscoe-

lasticity. This means the addition of a small amount of β-LG strengthened intermolecular

interactions (shear rheology) but exchange characteristics between the interface and the

bulk (dilational rheology) may have changed as well. In foam experiments especially at

low concentrations, synergistic effects in foam stability were detectable. But it also has to

be kept in mind that this effect probably is not solely because of increased shear viscoelas-

ticity but also because more surface active molecules are present in the system to occupy

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the interface. In addition, proteins are known to increase film thickness of foam lamellae

and to increase liquid content, which are both connected with longer foam stability.

When the β-LG concentration in the mixtures is increased both shear and dilational vis-

coelasticity decreased and had values similar to pure β-LG. This can be interpreted as a

reduction in intermolecular interactions (shear rheology) as well as may be attributed to

varying exchange characteristics (dilational rheology). This effect was not detectable in

foam experiments because foams of mixtures of QS and β-LG were still more stable than

pure β-LG. It has to be kept in mind that interfacial shear and dilational rheology can only

be indicators for the behavior of surface active molecules in real applications like foams.

Increased foaming ability and foam stability are attributed to high interfacial shear and di-

lational moduli but small changes in viscoelasticity do not necessarily affect foam

properties. Foams are complex and thermodynamically unstable systems and many factors

have to be taken into account to predict foaming and decay properties.

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II-6 Conclusion

In a binary system of Quillaja saponin and β-LG synergistic effects and interactions be-

tween both substances can occur. We analyzed various interfacial properties, like

adsorption behavior and shear and dilational rheology of mixtures of QS and β-LG to draw

conclusions to foam experiments. Fluorescence measurements on the interaction of QS and

β-LG in the bulk showed that it is highly likely that ground-state complexes are formed.

Whether or not these complexes change upon adsorption at the interface is subject to further

investigations. QS, β-LG and their mixtures form highly viscoelastic interfaces, which can

sufficiently resist dilational and shear deformation. Interfacial dilational and shear rheology

can provide crucial information on interfacial interactions and underlying interfacial phe-

nomena. It is assumed that intermolecular interactions between QS and β-LG at the

interface are either formed through hydrogen bonds and/or hydrophobic interactions. In

mixtures at low β-LG concentrations the interfacial properties are mainly governed by QS

but at higher β-LG concentrations the interfacial properties approach pure β-LG layers. The

ability of QS to build up and maintain stable foams is mainly caused by the strong viscoe-

lastic interfacial network, unlike common low-molecular-weight surfactants, which

stabilize by the Gibbs-Marangoni mechanism. Unlike in other surfactant/protein-systems

QS is not able to easily deplete β-LG from the interface once β-LG adsorbed.

We have here discussed differences between common surfactant/protein-systems and

the β-LG/QS-system. Although there have been studies on the interactions on Quillaja sap-

onins and other food proteins like lysozyme and β-casein, general knowledge on the

interactions of proteins and saponins is scarce. The combination of Quillaja saponins and

proteins is promising and should be analyzed further to benefit from this knowledge in food

products. Additional research on thin films for example with ellipsometry and Brewster-

angle microscopy can give further insights in foam lamella thickness and black film for-

mation. The formation of common and newton black films is attributed to very stable foam

lamellas, which lead to increased foam stability. As is well known, β-LG properties at the

oil/water-interface differ from those at the air/water-interface. It would therefore, be inter-

esting to analyze the interactions of β-LG and Quillaja saponin at the oil/water-interface to

correlate interfacial properties with emulsion stability. Furthermore, the finding of the in-

teractions between β-LG and QS at the air/water and oil/water-interface can be a basis to

analyze the properties of foams with oil content and their stability.

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Manuscript III

Mixtures of Quillaja saponin and beta-lacto-

globulin at the oil/water-interface:

Adsorption, interfacial rheology and emul-

sion properties

Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2017, V. 518,

pp. 46–56. doi: http://dx.doi.org/10.1016/j.colsurfa.2016.12.041.

Authors

Sandra Böttchera

Julia K. Kepplerb

Stephan Druscha

a Technische Universität Berlin, Institute for Food Technology and Food Chemistry

Department of Food Technology and Food Material Science

Königin-Luise-Str.22, 14195 Berlin

b Christian-Albrechts-Universität zu Kiel, Division of Food Technology, Institute of Hu-

man Nutrition and Food Science, Heinrich-Hecht Platz 10, 24118 Kiel, Germany

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III-1 Abstract

Aim of the present study was to investigate the interfacial properties of mixed films of

Quillaja saponins (QS) and beta-lactoglobulin (β-LG) at the oil/water-interface. It was hy-

pothesized that due to the differences in the physical characteristics of the dispersed phase

molecular interactions and film characteristics at the oil/water interface substantially differ

from the air/water-interface. Furthermore QS/β-LG-interactions will affect stability of

emulsions in a concentration-dependent manner.

Oscillating drop experiments were performed with subsequent analysis of raw data (Lis-

sajous-plots) to discover non-linear behavior upon compression and expansion. Interfacial

shear rheology as well as dynamic interfacial tension and two-fluid needle experiments

were performed to comprehensively characterize interfacial properties of mixtures of QS/β-

LG. Finally, emulsions were prepared and their stability, oil droplet size and ζ-potential

were determined.

It became obvious that QS dominates the interfacial film in a binary mixture as indicated

by dynamic interfacial tension and dilational rheology. Strain stiffening was observed for

mixed QS/β-LG interfacial layers upon dilational expansion at an amplitude above 2.8 %.

Intermolecular interactions increased in mixtures of QS/β-LG as indicated by shear rheol-

ogy. Emulsion experiments showed extensive aggregation of oil droplets in QS/β-LG-

emulsions with high content of β-LG. Aggregation of oil droplets increased velocity of

creaming and after 7 days a distinct creaming layer was visible. Changes in dispersity were

influenced by concentration and ratio of QS and β-LG.

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III-2 Introduction

Quillaja saponins (QS) are natural amphiphilic emulsifiers approved as a food additive in

the EU and US. Interfacial properties of saponins originate from the presence of hydrophilic

and hydrophobic parts in the molecule (Vincken et al. 2007). Saponins consist of a hydro-

phobic aglycone with hydrophilic chains of sugar attached to the aglycone. They may be

classified in two different groups depending on the number of chains. If a single sugar chain

is linked to the aglycone, typically at C-3, the resulting saponin is called ‘monodesmosidic’.

If an additional sugar chain is present, typically at C-28 (Güçlü-Üstündağ and Mazza 2007),

the structure is referred to as ‘bidemosidic’. Commercial available extracts from the Chil-

ean soap bark tree Quillaja saponaria Molina mainly consist of a mixture of various

bidesmosidic saponin derivatives (Thalhamer and Himmelsbach 2014; Bankefors et al.

2011; Bankefors et al. 2010).

Since QS is approved as a food additive, comprehensive studies on interfacial properties

(Wojciechowski 2013) and chemical constitution (Maier et al. 2015a) were published in

the past. The majority of studies on the interfacial properties of QS focused on the air/water-

interface. Several studies (Wojciechowski 2013; Golemanov et al. 2012; Stanimirova et al.

2011) determined interfacial rheology and adsorption kinetics. In addition, in one study

(the authors also proposed a model for interfacial arrangement of Quillaja saponin mole-

cules at the air/water-interface (Golemanov et al. 2012). In this model, a domain structure

was suggested, which explains the high elastic shear and dilational moduli of QS of

~80 mN/m and ~180 mN/m, respectively (Böttcher et al. 2016). High interfacial shear and

dilational moduli are rather unique for such small molecules like the saponins. Usually low-

molecular surfactants (<500 Da) do not form strong interfacial films and cannot withstand

shear and dilational deformation (Bos and van Vliet 2001). It was suggested that intermo-

lecular hydrogen bonds between neighboring sugar residues account for the strong

interfacial viscoelastic films (Golemanov et al. 2012).

However, it was also shown that the interfacial viscoelastic film of QS is much weaker

at the oil/water-interface. As discussed by Golemanov et al. (2014) the general opinion is

that interactions between the surface active molecules at the interface are reduced in the

presence of oil. There are two possible mechanisms to explain this phenomenon: Firstly,

oil may penetrate into the space between adsorbed surface active molecules or secondly,

surface active molecules are solubilized in the oil phase. In both cases the result is a less

dense packaging at the interface. It is assumed that the first mechanism occurs when using

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surfactants, which are not soluble in oil, and proteins. The second mechanism is relevant

for oil-soluble surfactants like non-ionic low-molecular weight surfactants. Golemanov et

al. (2014) showed that Quillaja saponins are not soluble in triglyceride-based oils. The au-

thors therefore concluded that the first mechanism applies and oil molecules penetrate

between adsorbed QS molecules thereby reducing intermolecular interactions.

Apart from fundamental studies dealing with interfacial rheology and adsorption, sev-

eral studies on practical aspects like formation and stability of QS-based emulsions were

published in recent years (Yang et al. 2013; Zhang et al. 2015, Zhang et al. 2016). Emul-

sions are per definition thermodynamically unstable. However, if stabilizing mechanism

sufficiently slow down thermodynamically driven changes in dispersity, emulsions are fre-

quently described as kinetically stable (Dunkhin et al. 2001). Using a shift in the oil droplet

size distribution as a marker, it was shown that QS-based emulsions are kinetically stable

for one month even at high storage temperatures up to 55°C (Yang et al. 2013). Stability

of QS emulsions was attributed to the highly negative ζ-potential at pH 7, which facilitates

electrostatic repulsion between oil droplets.

With respect to the use in more complex food matrices, investigation of the interaction

of QS with other food constituents is of utmost importance. The interaction between pro-

teins and low molecular weight surfactants has been subject of various studies in the past

and a summary goes beyond the scope of this introduction. In the bulk, complex formation

between surfactants, like sodium dodecyl sulfate and proteins was reported (Hu et al. 2011;

Kotsmar et al. 2009). Furthermore, it was shown that surfactants may penetrate into protein

films at the interface (Morris and Gunning 2008) and consequently may cause replace-

ment/desorption of proteins (Dan et al. 2015; Fainerman et al. 2006). The presence of

surfactant may substantially reduce interfacial shear and dilational viscoelastic moduli

(Dan et al. 2013, Ulaganathan et al. 2012; Maldonado-Valderrama and Patino 2010; Krägel

et al. 2008). More specifically with respect to QS, Piotrowski et al. (2012) analyzed inter-

actions of QS and β-lactoglobulin (β-LG) at the air/water-interface as well as at a

water/tetradecane-interface. The authors reported that dynamic interfacial tension is lower

in binary mixtures of QS and β-LG compared to only QS at the water/tetradecane-interface.

Our group recently showed in fluorescence experiments that QS and β-LG form a complex

in aqueous solution (Böttcher et al. 2016). Furthermore, synergistic effects with respect to

the complex viscoelastic moduli of interfacial films at the air/water-interface and with re-

spect to foam stability were found. It was hypothesized that intermolecular hydrogen bonds

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between β-LG and sugar residues of QS are formed, which account for the increased vis-

coelasticity.

The question remains whether mixtures of QS and β-LG also form viscoelastic films at

the oil/water-interface and how film properties affect the stability of emulsions. There are

substantial differences in the adsorption of proteins at the oil/water-interface compared to

the air/water-interface in general, and for β-LG in particular (Zare et al. 2016). As recently

reviewed by Zhai et al. (2013) a significant increase in understanding of the unfolding and

molecular mechanisms of adsorption of β -LG was achieved in the last couple of years but

still the process is not fully understood, yet. The main driving force for the unfolding of β-

LG at the interface is the re-orientation of the hydrophobic sites of the molecule towards

the oil phase. To characterize the conformation of β-LG at the oil/water-interface, different

techniques were used in the past, like Fourier transform infrared (FTIR) spectroscopy, far-

UV circular dichroism (CD) spectroscopy and synchrotron radiation circular dichroism

(SRCD). β-LG looses its tertiary structure upon adsorption, but the secondary structure

including α-helices and β-sheets are preserved to a great extent depending on the hydro-

phobicity of the oil phase. It was concluded that structural rearrangements are more

pronounced at the less polar interface n-alkane/water (e.g. tetradecane) than at more polar

interface triglyceride/water.

Based on the considerations above it must be assumed that interactions between QS and

β-LG at the oil/water-interface significantly differ from those at the air/water-interface

(Maldonado-Valderrama and Patino 2010). Aim of the present study therefore was to char-

acterize the interaction between QS and native β-LG at the MCT-oil/water-interface with

respect to adsorption and interfacial rheology. In a second part, emulsions stabilized by

blends of QS and native β-LG are prepared to evaluate the impact of interfacial interactions

on emulsion properties. Interfacial properties of binary mixtures and the individual sub-

stances are determined using dynamic two-fluid needle analysis, dynamic interfacial

tension measurement as well as dilational and shear rheology for characterization of the

interfacial film. Interfacial microstructure in dilational experiments is qualitatively and

quantitatively characterized by Lissajous-plots. In addition, emulsification trials are per-

formed for the individual constituents and binary mixtures using high-pressure

homogenization. Emulsions are characterized with respect to oil droplet size, ζ-potential

and emulsion stability after seven days.

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III-3 Material and methods

III-3.1 Materials

In the present study, a commercial saponin extract from the bark of the soap bark tree Quil-

laja saponaria Molina (QS) was used, which was kindly provided by Ingredion Germany

GmbH (Hamburg, Germany). Solid phase extraction on a C-18 column (10 g/70 mL

Thermo Scientific) was used to purify the saponin extract, as described in Böttcher et al.

(2016). As Keppler et al. (2014a) described, native undenaturated β-Lactoglobulin (β-LG)

was isolated from whey protein isolate (Fonterra DSE 6668). Medium-chain triglyceride

oil (MCT-oil) WITARIX® MCT 60/40 was purchased from CREMER OLEO GmbH &

Co. KG (Hamburg, Germany). Surface active constituents in the MCT-oil were removed

through adsorption onto magnesium silicate (Florisil®, Sigma-Aldrich GmbH, Seelze, Ger-

many).

All aqueous solutions were prepared in 10 mM potassium phosphate bfuffer (pH 7) and

continuously stirred at room temperature for at least 8 h. For all experiments QS concen-

tration was fixed at 0.005 %wt. Four different concentrations of β-LG were chosen: 0.005,

0.01, 0.05 and 0.1 %wt. The low β-LG-concentration of 0.005 and 0.01 % as well as their

mixtures with QS will be referred to as ‘β-LGlow‘ and ‘QS/β-LGlow‘, respectively. In the

same manner the high β-LG-concentration of 0.05 and 0.1 % will be referred to as ‘β-

LGhigh’ and the mixture with QS as ‘QS/β-LGhigh’ throughout the whole manuscript.

III-3.2 Drop shape analysis of adsorption, dynamic interfacial tension and dilational

rheology

Dynamic interfacial tension was measured by pendant drop tensiometry (contact angle me-

ter OCA-20, DataPhysics Instruments GmbH, Filderstadt, Germany). MCT-oil was poured

in a glass cuvette and a droplet of surfactant solution (28 µL) was generated through a

needle immersed into the MCT-oil. Drop shape was recorded with a frame rate of five

frames per minute for 20 min. Interfacial tension was calculated by fitting the Young-La-

Place-equation to the drop shape according to Najmabadi et al. (2013).

Interfacial dilational rheology was studied using the above mentioned contact angle me-

ter equipped with an oscillation unit ODG-20 and a slightly modified method as reported

by Tamm & Drusch (2017). Briefly, a droplet of 32 µL of the sample solution was manually

generated in MCT-oil and allowed to equilibrate for 16 h. After equilibration an amplitude

sweep ranging from 1.4 to 10 % deformation amplitude was performed at a frequency of

0.1 Hz. The interfacial dilational modulus E* was calculated from the oscillation cycles.

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The elastic modulus (storage modulus, E’) and viscous modulus (loss modulus, E’’) were

derived from E*. The phase angle Φ, which results from changes in interfacial tension due

to area change, is calculated using tan Φ=E’’/E’. At small Φ (<45°) the interface is pre-

dominantly elastic (E’>E’’), whereas at high Φ (>45°) the interface shows more viscous

(E’<E’’) properties.

To get further insights in interfacial microstructure Lissajous-plots of the amplitude

sweep were analyzed. Therefore, the change in interfacial tension described via the surface

pressure Π=σ-σ0 (stress) was plotted against the area change ΔA/A0 with ΔA=A-A0 (strain)

for different amplitudes. A0 represents the interfacial area at strain 0 and A the area at a

certain strain. A Lissajous-plot is considered as a fingerprint of an interface, which shows

the response of the interfacial layer to compression and expansion (Sagis and Fischer 2014).

Data were quantified by calculating the strain-stiffening ratio S as defined by Ewoldt et al.

(2008) and extended by van Kempen et al. (2013) for dilational experiments. Dilational

extension is described by Sext=(ELE-EME)/ELE and compression by Scom=(ELC-EMC)/ELC,

where EL is the large strain modulus and EM is the minimum strain modulus. The subscripts

‘E’ and ‘C’ refer to extension and compression, respectively. All variables are slopes de-

rived from the Lissajous-plot, where EM is obtained at a strain of 0 and EL at maximum

strain. A detailed description and visualization of the method was published by van

Kempen et al. (2013). Interpretation of S is as follows: at S=0 the interfacial layer shows

either linear elastic or linear viscoelastic behavior. The latter is characterised by an elliptic

shape of the Lissajous-plots (widening between compression and expansion curve). When

S≠0 the interfacial layer has non-linear viscoelastic properties. Furthermore, S<0 indicates

non-linear behavior due to strain-softening and at S>0 the reason is strain-stiffening of the

interfacial layer upon compression or expansion.

For experiments on adsorption a two-fluid needle method developed in our group was

used as it has previously been reported (Böttcher et al. 2016). Briefly, a droplet of buffer

solution (28 µL) was generated at the tip of a needle with an inner diameter of 1.65 mm. A

second needle with an inner diameter of 0.5 mm is inserted within the first needle and a

drop of surfactant solution (2 µl) is injected into the buffer droplet using the dosing unit of

the contact angle meter. Movement of the emulsifier through the droplet is characterized

by the so-called lag-time, i.e. the time until surface pressure for the first time differs from

zero. It must be emphasized that this movement must not be interpreted as diffusion. Ad-

sorption from the subsurface (liquid layer adjacent to the interface, Fainerman et al. 2001)

to the interface is described by Π2s).

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III-3.3 Interfacial shear rheology at the oil/water-interface

Shear experiments was performed using a Physica MCR301 rheometer (Anton Paar Ger-

many GmbH, Ostfildern, Germany) equipped with a biconus for interfacial rheology. An

aliquot of 120 mL of the individual sample solution was poured into a cell and bubbles

were removed from the interface. Afterwards the biconus was placed directly at the inter-

face and covered with 100 mL purified MCT-oil. Film formation was monitored by

applying a strain amplitude γ0 of 0.1 % and frequency f of 1 Hz to the interfacial film every

5 min. Samples were measured at 25°C. In a similar manner as described for the dilational

rheology, viscoelastic properties of the interface may be described as complex shear mod-

ulus G*, the elastic modulus (G’) and viscous modulus (G’’).

III-3.4 Emulsification and emulsion stability

Oi-in-water emulsions were prepared in two subsequent steps. At first, solutions of QS, β-

LG and mixtures of both constituents were prepared 16 h prior to emulsification, 20 % pu-

rified MCT-oil containing 0.01 % oil red O (a hydrophobic dye) was added and a coarse

emulsion was prepared by using a rotor-stator system (Ultra-Turrax T25 basic, IKA -Werke

GmbH & CO. KG, Staufen, Germany) at 13,500 rpm for 30 s. In the second step, the coarse

emulsion was homogenized in a high-pressure homogenizer (Panda 2K, Niro Soavi

Deutschland, Lübeck, Germany) at 600 bar with 4 passes. An aliquot of the emulsions was

transferred into graduated test tubes. Stability was checked after 7 days both, visually as

well as via light microscopy at 400-fold magnification.

III-3.5 Determination of oil droplet size distribution and ζ-potential

The oil droplet size distribution was determined by static light scattering (Horiba LA-950,

Retsch Technology GmbH, Haan, Germany). A droplet of the emulsion was diluted in

mono-distilled water. The pump of the measuring device was set to 8 and the stirring unit

to 3. A refractive index of 1.45 was used for the calculation for all samples. The oil droplet

size distribution is reported based on the volume distribution. Different percentiles of the

oil droplet size distribution (d10, d25, d50, d75 and d90) are displayed as a box plot to facilitate

comparison between the samples. The ζ-potential was determined using a Nano Zetasizer

ZS from Malvern Instruments GmbH (Herrenberg, Germany). Emulsions were diluted

1:100 and measured in clear disposable cells (DTS 1060 C, Malvern Instruments GmbH).

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III-4 Results

III-4.1 Adsorption and dynamic interfacial tension at the oil/water-interface as deter-

mined by drop shape analysis

Movement of QS through the droplet was very fast as indicated by a short lag-time of 2.8 s.

In addition, surface pressure increased fast as shown by the Π2s-value of 12.9 mN/m (Figure

III-1). In contrast, β-LG showed a rather long lag-time and surface pressure was compara-

bly low after 2 s (low Π2s-values). For β-LG, with increasing concentration lag-time

decreased from 34.4 s to 6.3 s and surface pressure (Π2s) increased from 2.7 to 7.8 mN/m.

Adsorption from the subsurface to the oil-water interface of mixtures of QS/β-LG (as indi-

cated by the surface pressure Π2s) was dominated by QS. In addition, movement of the

binary mixture of QS/β-LG through the droplet (described by the lag-time) was distinc-

tively slowed down in the presence of β-LG compared to the lag- time of QS.

Figure III-1 Results of A) lag-time and B) surface pressure after 2 s of adsorption (Π2s) in rela-

tion to the β-LG concentration of 0.005 % QS (◇), β-LG (○) and mixtures of

0.005 % QS and β-LG (⚫) at the MCT-oil/water-interface

0,001 0,01 0,1

Π2s [mN/m]

Concentration β-LG [%]

0.01

0.1

0.005 %

0,001 0,1

Lag-time [s]

Concentration β-LG [%]

0.005 %

0.01

0.1

(A)

(B)

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As presented in Table III-1 QS efficiently lowered the dynamic interfacial tension after 20

min (12.6 mN/m) to a lower value than even the highest β-LG concentration (13.7 mN/m).

In mixtures of QS/β-LG interfacial tension was slightly lower than for individual QS or β-

LG samples.

Table III-1 Dynamic interfacial tension (IFT) of β-LG-solutions and β-LG/QS-mixtures meas-

ured for 20 min with pendant drop analysis at the oil/water-interface

cQS [%wt] cBLG [%wt] β-LG/QS [%wt/%wt] IFT [mN/m]

0.005 15.6 ± 0.1

0.01 14.4 ± 0.1

0.05 13.6 ± 0.0

0.1 13.7 ± 0.0

0.005

0 12.6 ± 0.1

0.005 1 12.7 ± 0.1

0.01 2 12.4 ± 0.1

0.05 10 12.1 ± 0.1

0.1 20 12.0 ± 0.0

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III-4.2 Dilational rheology determined by droplet oscillation and analysis of non-lin-

ear phenomena of interfacial layers

Via compression and expansion experiments (dilational rheology) it is possible to

characterize the interfacial layer with respect to interfacial rearrangements and exchange

processes between the bulk and the interface (Miller et al. 1996; Freer et al. 2004). The

interfacial layer of QS showed a lower interfacial dilational modulus E* and was more

viscous than interfacial layers consisting of β-LG. Mixed interfacial layers of QS/β-LG

were rather viscous, which was similar to interfaces of QS, but E* increased with increasing

β-LG-concentration. When comparing the interfacial dilational modulus E* (see Figure III-

2) it becomes obvious that QS had a very low E* of 15 mN/m. With increasing β-LG-

concentration E* increased from 51 to 68 mN/m. In mixtures of QS/β-LG with up to

35 mN/m, E* was lower than E* of an interface solely occupied by β-LG.

Figure III-2 Interfacial dilational modulus E* in relation to β-LG-concentration determined by

dilational oscillation at f=0.1 Hz, 2.8 % amplitude of 0.005 % QS (◇), β-LG (○) and

mixtures of 0.005 % QS and β-LG (●) at the MCT-oil/water interface. Error bars rep-

resent 10 % deviation, which was determined in previous experiments.

No distinctive reduction in E* was found with increasing amplitude (data not shown) indi-

cating that the interfacial layer was still intact and may only rupture at higher amplitudes

as reported by Wan et al. (2016). In Figure III-3 the phase angle Φ is displayed versus the

deformation amplitude ΔA/A0. The phase angle was very similar for all β-LG samples in-

dependent from the concentration. The low value of approximately 7° indicates a highly

elastic interfacial film. In contrast with approximately 40°, the phase angle of QS was rather

0,001 0,1

E* [mN/m]

Concentration β-LG [%]

0.005 %

0.01

0.1

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high. The interfacial film still is predominantly elastic, but also has a high viscous modulus.

Mixed interfacial films consisting of QS/β-LG also showed a high phase angle, but with

increasing β-LG concentration, Φ decreased (elastic properties were higher).

Figure III-3 Phase angle Φ in relation to area change ΔA/A0 determined by dilational oscillation at

f=0.1 Hz at the MCT-oil/water interface of 0.005 % QS (+), 0.005 % β-LG (◇),

0.01 % β-LG (△), 0.05 % β-LG (○), 0.1 % β-LG (□); filled symbols indicate β-LG

is mixed with 0.005 % QS

In Figure III-4 the Lissajous-plots of QS, β-LG and mixtures of both are displayed at dif-

ferent amplitudes. Lissajous-plots were elliptic and thus showed a linear-viscoelastic

behavior for QS and β-LG as well as their mixtures at low amplitude. Non-linear behavior

in form of strain-stiffening, was detected in mixed interfacial films consisting of QS/β-LG-

at a high amplitude. At the lowest amplitude (1.4 %) random scattering occurred and neg-

atively affected the interpretation of the graphs for all samples. At an intermediate

amplitude of 2.8 % the plots became more meaningful and all Lissajous plots showed an

elliptic shape when oscillated. At a high amplitude (4.2 and 7.0 %) all graphs except β-LG

revealed an asymmetric shape with strain-stiffening behavior upon expansion. There were

marked differences between the sinusoidal response of β-LG, QS and the mixed interfacial

layers. When interfacial layers with QS and mixtures of QS/β-LG were oscillated a widen-

ing between compression and expansion curve occurred, which was absent in β-LG layers.

In mixed interfacial films of QS/β-LG non-linearity decreased with increasing β-LG-con-

centration and the shape of the Lissajous plot approached the form of the plot of β-LG.

110

Φ[°]

ΔA/A0 [%]

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Figure III-4 Interfacial tension σ versus area change ΔA/A0 (Lissajous-plots) determined by dila-

tional oscillation at f=0.1 Hz at different amplitudes 1.4, 2.8, 4.2 and 7.0 % at the

MCT-oil/water interface of a) 0.005 % QS (+), b) 0.01 % β-LG (△), c) 0.005 %

QS+0.005 % β-LG (◆), d) 0.005 % QS+0.05 % β-LG (△), e) 0.005 % QS+0.05 %

β-LG (●), f) 0.005 % QS+0.1 % β-LG (□)

σ[mN/m]

-2

-2 0 2

-2

-2 0 2

-2

-3 0 3

-4

-5 0 5

-2

-2 0 2

-2

-2 0 2

-2

-3 0 3

-4

-5 0 5

-2

-2 02

-2

-2 -1 012

-2

-3 0 3

-4

-5 0 5

-2

-2 0 2

-2

-2 0 2

-2

-3 0 3

-4

-5 0 5

-2

-2 0 2

-2

-2 0 2

-2

-3 0 3

-4

-5 05

-2

-2 0 2

-2

-2 0 2

-2

-3 0 3

-4

-5 0 5

ΔA/A

[%]

amplitude

[%]

4.2 7.01.4 2.8

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In Figure III-5 the evolution of the S-factor versus the amplitude is shown. No difference

of the S-factor was found upon compression but substantial differences occurred upon ex-

pansion. At an amplitude higher than 4.2 % the S-factor for QS and mixtures of QS/β-LG

distinctively increased. There was a concentration dependence of the S-factor in mixtures

of QS/β-LG: at low β-LG-concentration the increase of the S-factor at high amplitude was

more pronounced than at high β-LG-concentration.

Figure III-5 S-factor versus amplitude during expansion (left panel) and compression (right

panel) of a droplet at the oil/water-interface determined by dilational oscillation at

f=0.1 Hz of 0.005 % QS (+),0.005 % β-LG (◇), 0.01 % β-LG (△), 0.05 % β-LG

(○), 0.1 % β-LG (□); filled symbols indicate β-LG is mixed with 0.005 % QS

-0,5

0,0

0,5

1,0

110

S [-]

Amplitude [%]

1.0

0.5

0.0

-0.5

expansion

110

Amplitude [%]

compression

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III-4.3 Shear rheology of interfacial layers

Experiments on shear rheology give insights in lateral intermolecular interactions between

molecules. Results of shear rheological experiments supported the results from dilational

rheological experiments. In Figure III-6 the complex (G*), elastic (G’) and viscous (G’’)

moduli of all samples are presented. The interfacial layer of QS showed a very small G* of

7.4 mN/m with a high viscous modulus G’’ of 4.9 mN/m. G* of the interfacial layer of β-

LG and binary mixtures of QS/β-LG as well as the viscous contribution increased with

increasing β-LG-concentration. For the highest β-LG-concentration in mixtures of QS/β-

LG ratio of G’’/G’ was 0.56 in contrast to interfacial layers solely prepared with β-LG,

which had a ratio of G’’/G’ of 0.30.

Figure III-6 Complex (G*), elastic (G’) and viscous (G’’) shear moduli for 0.005 % QS; 0.005,

0.01, 0.05 & 0.1 % β-LG and 0.005 % QS mixed with 0.005, 0.01, 0.05 & 0.1 % β-

LG (from left to right) at the MCT-oil/water-interface measured with f=1 Hz, de-

formation=0.1% after 9 h of film formation

G*, G', G'' [mN/m]

G''

0.1

0.05

0.01

0.005

0.1

0.05

0.01

0.005

QS+β-LG

high

QS+β-LG

low

β-LG

high

β-LG

low

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III-4.4 Oil droplet size distribution of emulsions as determined by static light

scattering

The emulsion prepared with QS exhibited a narrow oil droplet size distribution with a low

median (d50). In contrast, emulsions stabilized with β-LG had a comparably broad oil drop-

let size distribution and a high d50. In mixtures of QS/β-LG the d50 decreased with

increasing β-LG-concentration and led to distinctively smaller oil droplets compared to

emulsions made of QS. In Figure III-7 the oil droplet size distribution for all samples is

summarized. The median of the oil droplet size distribution for β-LG-stabilized emulsions

ranged from 21.3 to 9.0 µm for a β-LG concentration of 0.005 and 0.1 %, respectively. The

median of the oil droplet size distribution of the QS-emulsion was markedly lower with a

d50 of 4.1 µm. In binary mixtures of QS/β-LG with a β-LG concentration of 0.005 % the

median was 2.5 µm and in mixtures with 0.1 % β-LG the median was reduced to 0.6 µm.

Figure III-7 Oil droplet size of emulsions prepared with 0.005 % QS (1st boxplot), 0.005…0.1 %

β-LG (2nd to 5th boxplot) and mixtures of 0.005 % QS and 0.005…0.1 % β-LG (6th

to 9th boxplot) with upper whisker representing d90, upper box end d75, dash in the

box d50, lower box end d25 and lower whisker d10

0,1

1,0

10,0

100,0

QS 0.005 BLG

0.005

BLG 0.01 BLG 0.05 BLG 0.1 QS+BLG

0.005

QS+BLG

0.01

QS+BLG

0.05

QS+BLG

0.1

Oil droplet size [µm]

102

101

100

10-1

QS β-LGlow

0.01

0.005 0.05 0.1

0.005

QS/β-LGlow

0.1

0.05

0.01

0.005

β-LGhigh QS/β-LGhigh

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III-4.5 ζ-potential of the emulsion droplets

In binary mixtures of the two emulsifiers at high β-LG-concentration (QS/β-LGhigh) the ζ-

potential was similar to emulsions stabilized with β-LG. In contrast, in emulsions stabilized

with binary mixtures with low β-LG concentration (QS/β-LGlow) the ζ-potential was dom-

inated by QS. In Figure III-8 the ζ-potential of emulsions with QS, β-LG and mixtures of

QS/β-LG are shown. The ζ-potential of all emulsions in the present study was lower than -

30 mV. With increasing β-LG concentration the ζ-potential decreased from -51.1 to -

67.1 mV for emulsions with 0.005 % to 0.1 % β-LG, respectively.

Figure III-8 ζ-potential in relation to the β-LG concentration of emulsions (600 bar, 4 passes,

5 % MCT-oil) with 0.005 % QS (◇), β-LG (○) and mixtures of 0.005 % QS and β-

LG (●)

-75

-50

-25

0,001 0,01 0,1

ζ [mV]

Concentration BLG [%]

QS 0.005 %

0.01

0.1

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III-4.6 Visual analysis of emulsion stability after a storage time of 7 days

Emulsion stability was evaluated visually as presented in Figure III-9. Different kinds of

destabilization mechanisms were observed in the analyzed emulsions. Coalescence is indi-

cated by a continuous red oil layer on top of a serum layer. In contrast, creaming is

characterized by color differences between upper and lower phase without clear phase sep-

aration and thus varying difference in color intensity. In the emulsion prepared with QS,

destabilization was mainly due to creaming and to a small portion caused by coalescence.

Similar phase separation by creaming was observed for β-LGhigh-emulsions. In contrast, β-

LGlow and QS/β-LGlow-emulsions were mainly destabilized by coalescence.

Figure III-9 Photographic images of emulsions prepared with 0.005 % QS; 0.005, 0.01, 0.05 &

0.1 % β-LG and 0.005 % QS mixed with 0.005, 0.01, 0.05 & 0.1 % β-LG (from left to right) after

7 days of storage

β-LG

high

β-LG

low

QS/β-LG

high

QS/β-LG

low

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III-5 Discussion

In the first part of the discussion the interfacial properties of binary mixtures of QS and β-

LG at the oil/water-interface are compared with the corresponding properties of the indi-

vidual constituents. Afterwards, results of emulsion experiments are discussed with

particular focus on emulsion stability.

Based on the model of Ward and Tordai (1946) the time dependence of the interfacial

tension of solutions on a time-scale of a few seconds was analyzed in two-fluid needle

experiments. Data are presented as time interval until surface pressure starts to increase

(lag-time) and surface pressure after two seconds (Π2s). The lag-time of QS was low and

surface pressure after 2 s was high as it is generally expected for a low molecular weight

emulsifier. QS (~2 kDa) is smaller than β-LG and therefore shows an increased mobility in

the aqueous environment and adsorbs from the subsurface to the interface (see Figure III-

1). In comparison, β-LG (18 kDa) exhibited a slower movement through the droplet and a

lower surface pressure. These results reflect the typical behavior of globular proteins, where

adsorption expressed as surface pressure is retarded due to slow adsorption kinetics and

unfolding. The surface pressure after 2 s of mixtures of QS/β-LG was similar to QS, but

the lag-time was distinctively higher than the lag-time of QS. In a previous publication, we

postulated (based on fluorescence experiments) a complex formation between β-LG and

QS (Böttcher et al. 2016). By performing fluorescence experiments it is possible to deter-

mine non-covalent binding sites of β-LG. As Keppler et al. (2014b) reported it is advisable

to use the Cogan-Plot for analyzing the fluorescence results because the parameter ‘maxi-

mum number of binding sites’ is relatively stable compared to other methods. As reported

in the previous paper a maximum of 1.7 binding sites was determined between QS and β-

LG, which can be derived from the inverse slope of the Cogan-Plot (see Figure III-10).

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Figure III-10 Fluorescence measurements on the interactions of β-LG and QS showing results of

the Cogan-Plot with P=β-LG-concentration, α=fraction of free binding sites and

Lt=QS-concentration. Adapted from Böttcher et al. (2015) with permission from

Elsevier.

It is possible that movement through the droplet is slowed down by a size increase due to

this complex formation. Although QS has only 10 % of the molecular mass of β-LG the

hydrodynamic radii are quite similar. The hydrodynamic radii were reported before as

1.5 nm (Böttcher und Drusch 2016) and 2.5 nm (Taulier and Chalikian 2001) for QS and

β-LG, respectively. It is more likely that the amount of free QS molecules is reduced be-

cause QS molecules reversibly bind to β-LG or movement is slowed down by steric

hindrances. As shown before for the adsorption at the air-water interface (Böttcher et al.

2016), these complexes formed in the bulk do not influence the adsorption from the sub-

surface as indicated by a similar surface pressure for QS and mixtures of QS/β-LG in the

present study.

In addition, it needs to be discussed why in two fluid needle experiments on interfacial

adsorption differences in lag-time between QS and binary mixtures of QS/β-LG at the

oil/water-interface did not occur when analyzing the lag-time at the air/water-interface

(Böttcher et al. 2016). Complex formation occurs in the bulk of the aqueous phase and

therefore it is unlikely that the formation is affected by the type of hydrophobic phase. It is

rather assumed that differences in lag-time were not large enough to unambiguously differ-

entiate between QS and mixtures of QS/β-LG in the experimental setup at the air/water-

interface. Lag time was generally lower in the cited study and thus a “lower resolution”

occurred, which may be attributed to a smaller droplet volume of 14 µL in the study on the

y = 0.59x -4E-07

R² = 0.94

0,E+00

1,E-05

2,E-05

0,0 0,0 0,0

P*α

Lt*α/(1-α)

10-5 2∙10-

-5

2∙10

-5

4∙10-7

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adsorption behavior at the air/water-interface compared to 28 µL in the present study. A

large droplet diameter in the experimental setup is only possible when the hydrophobic

phase sufficiently increases buoyancy of the droplet. In contrast, a small droplet diameter

results in a reduced distance from the inside of the droplet to the interface and thus a reduc-

tion in lag-time.

Upon prolonged measuring time, QS still dominated interfacial tension in mixtures of

QS and β-LG as indicated by results of dynamic interfacial tension (see Table III-1). The

results are in agreement with data from Piotrowski et al. (2012), who reported only slightly

lower values for the dynamic interfacial tension in mixtures of QS/β-LG in comparison to

QS.

In dilational and shear experiments interfacial layers of β-LG showed more resistance

against deformation (as indicated by high values for the complex moduli E* and G*) with

a higher elastic modulus in comparison to an interfacial QS layer (see Figure III-2 & Figure

III-6). Mixed interfacial layers of QS and β-LG were less viscoelastic (lower complex mod-

uli) at the oil/water-interface compared to the air/water-interface (Böttcher et al. 2016). In

the present study interfacial layers from binary mixtures of QS/β-LG exhibited high viscous

moduli (E’’ and G’’) resulting in relatively high Φ and tan δ similar to QS.

Lissajous-plots were analyzed to get more insights into the rheological behavior of in-

terfaces. The use of Lissajous-plots as an instrument for rheological characterization of

interfacial films subjected to dilational stress is rather new in food-oriented disciplines.

Lissajous-plots may be used to determine the viscoelastic regime (Sagis and Fischer 2014)

and/or microstructure of interfacial films (Wan et al. 2016).

In general, Lissajous-plots illustrate the stress (interfacial tension) versus strain (area

change) for the oscillation cycles. Within these plots viscoelastic behavior during expan-

sion and compression is distinguished. In a linear regime, interfacial tension oscillates

sinusoidal over time when low amplitudes/area changes are applied. A symmetrical diverge

in the Lissajous-plot (widening between compression and expansion curve) indicates vis-

cous properties of the interface (Sagis and Fischer 2014). When area changes become too

high, non-linear behavior may occur. Non-linear behavior is reflected by non-elliptic curves

upon compression and/or expansion. The response of the interface becomes irregular and

varies distinctively from the sinusoidal form. However, dilational moduli are still calculated

from raw data by Fourier transformation by most programs regardless of non-linear visco-

elastic behavior.

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All samples showed linear viscoelastic behavior upon compression even at high ampli-

tude, which may be deduced from a linear decrease in the Lissajous-plots and an S-factor

equal to zero (see Figure III-3 & Figure III-5). During expansion clear differences between

samples were visible. Expansion of β-LG-interfaces at low and high amplitudes did not

result in a change in the shape of the Lissajous-plot or the S-factor. When a QS-stabilized

interface was subject of a sinusoidal deformation, beginning of strain stiffening was detect-

able upon expansion at higher amplitude as indicated by S>0. For mixtures of QS/β-LG a

marked increase of the S-factor upon expansion was determined with increasing amplitude,

which indicates strain stiffening like in QS-layers. Mixed interfacial layers of QS/β-LG

generally showed a similar widening between compression and expansion curves as QS

(see Figure III-4), which indicates viscoelastic behavior. However, in contrast to QS the

response of the mixed films was non-linear. At the beginning of the expansion a steep in-

crease (lower left quadrant) of the interfacial tension with only small change in interfacial

area was detected, which shows that the interfacial network initially restrains the area ex-

pansion (but not the volume expansion). The interfacial network opposes the area

expansion but because additional solution is pumped into the droplet the droplet shape is

changing into a more spherical shape. It can be hypothesized that the mixed film cannot

rapidly react to the increase in volume (expansion) because of increase stiffness. This might

be due to an increased cohesion/interaction between the molecules, which may be caused

by prior compression. From this observation it can be concluded that the interfacial pack-

aging of β-LG is distinctively changed by the presence of QS.

After discussing interfacial properties we focus on stability of emulsions prepared with

QS, β-LG and their mixtures in the second part of the discussion. Emulsion stability is a

complex matter where various destabilization phenomena, like creaming, Ostwald-Ripen-

ing, coalescence and aggregation may simultaneously happen (Walstra 2003). As described

by Stokes’ law, velocity of creaming of oil droplets increases with an increase in the dif-

ference in density between continuous and dispersed phase or oil droplet size, and decreases

with increasing viscosity of the continuous phase. Intermolecular interactions resulting

from attractive van der Waals forces and (in the case of two oil droplets in a macro-emul-

sion) repulsive electrostatic forces affect aggregation as well as steric hindrance (in the case

of high molecular weight emulsifiers). Both may prevent aggregation and coalescence of

oil droplets. As reviewed by Heurtault et al. (2003) with increasing ζ-potential electrostatic

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repulsion between droplets increases, which is one factor preventing aggregation of oil

droplets. In general, emulsions with an absolute ζ-potential below 30 mV may be prone to

aggregation due to insufficient electrostatic repulsion. In contrast, an absolute ζ-potential

higher than 60 mV is considered ideal and is associated with long-term kinetic stability as

a result of high electrostatic repulsion. The ζ-potential of all emulsions in the present study

was lower than -30 mV, indicating that electrostatic repulsion will support kinetic stability

of the samples (see Figure III-8).

In general, use of QS as an emulsifier results in emulsions with sufficiently small oil

droplets as shown in different studies on saponin-based nanoemulsions (Bai et al. 2016;

Kaur et al. 2016). However, in the present study a concentration of QS was chosen, that

did not yield emulsions with long-term kinetic stability. This was done on purpose to ensure

that positive effects on droplet size reduction and/or kinetic stability by addition of β-LG

become obvious. As a consequence, the mean oil droplet size of the QS-emulsion was ra-

ther large and emulsions were prone to creaming.

Net repulsion as indicated by the ζ-potential is usually considered to prevent aggrega-

tion. A negative ζ-potential (-30 mV) of QS emulsions was reported before by several

authors and was attributed to the carboxylic group in the molecular structure of Quillaja

saponin (Yang et al. 2013; Zhang et al. 2016; Maier et al. 2015b). Zhang et al. (2016) used

a different Quillaja saponin extract but reported similar ζ-potential values of about -30 mV

at pH 7. Using a similar QS extract like in the present study, Yang et al. (2013) reported an

even lower ζ-potential for emulsions prepared with QS of -70 mV at pH 7. Differences in

ζ-potential may be linked to the purification process applied in the present study. Emulsions

prepared with crude, non-purified saponin extract had a ζ-potential of -50 mV at pH 7 (re-

sults not shown). It is clear that the ζ-potential of QS extracts is not only caused by saponin

molecules but to a high extent by negatively charged organic compounds other than sapo-

nins in the extract. These compounds may adsorb at the interface, which influences net

charge of the oil droplets. Phenols may be present at the droplet interface because of inter-

actions between adsorbed phenolic compounds and saponins. As Tippel et al. (2017)

showed, hydrophilic phenolic compounds are partially removed during purification, but a

certain proportion is still present. These anionic residues are phenolic compounds like (+)-

piscidic acid, syringic acid and p-coumaric acid (Maier et al. 2015a; Tippel et al. 2017).

However, since the QS content in the emulsions was below the CMC and not sufficient

to completely cover the droplet surface, electrostatic repulsion did not prevent aggregation.

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In general, a Weber number may be calculated by comparing external stress in the creaming

layer against internal stress given as the Laplace pressure, and one may decide, whether

drops remain unformed or if a flat film between them will be formed. Close proximity of

oil droplets, i.e. a thin film between close droplets, generally facilitates film rupture and

may lead to coalescence as partially observed in the present study. A gradient in interfacial

tension occurs as a consequence of a liquid flow during film-thinning and film rupture may

occur through hole formation in the interfacial film and development of capillary waves.

The latter are damped through intermolecular electrostatic repulsion and slow down coa-

lescence.

Oil droplet size of β-LGlow-emulsions was rather high and oil droplets showed a negative

ζ-potential. A large oil droplet size increases velocity of creaming as stated by Stokes’ law.

Although oil droplets had a negative ζ-potential of approximately -60 mV, rapid coales-

cence and subsequently complete phase separation (indicated by red top layer in Figure III-9)

was observed. The reason again is an insufficient concentration of the emulsifier (here: β-

LG). Large oil droplets increase the effective film radius upon aggregation. In combination

with an insufficient protein load at the interface coalescence may rapidly occur. At high

protein concentration (β-LGhigh) coalescence was reduced due to a higher interfacial con-

centration at the interface, but creaming still occurred due to the large oil droplet size.

Microscopic images showed no aggregation of oil droplets after 7 days of storage, which

would occur upon bridging flocculation.

QS/β-LGlow-emulsions showed creaming and partial coalescence, but no complete phase

separation occurred (see Figure III-9). Although a higher content of emulsifier was present,

QS/β-LGlow-emulsions were more prone to coalescence than the QS-emulsion. But at the

same time coalescence was less pronounced compared to β-LGlow. Interactions between QS

and β-LG in bulk and at the interface and displacement of β-LG by QS have been described

before (Böttcher et al. 2016). Non-covalent interactions between β-LG and QS (as reported

for other β-LG systems) may change the tertiary and quaternary structure of the protein, as

well as its structural flexibility, thereby altering the surface activity of the complex

(Staszewski et al. 2014). However, the kind of interaction was not determined yet and it

has to be kept in mind that β-LG undergoes structural changes upon adsorption at the

oil/water-interface, which may alter the interactions between QS and β-LG (Dan et al.

2013). It can therefore be concluded that changes in dispersity may be attributed to inter-

actions between QS and β-LG, which increases coalescence.

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Visual appearance and stability of QS/β-LGhigh-emulsions was similar to β-LGhigh-emul-

sions although oil droplet size in QS/β-LGhigh-emulsions was sufficiently smaller (see

Figure III-7) and ζ-potential was very low. We therefore took microscopic images of all

emulsions after one week and detected a large number of aggregates in QS/β-LGhigh (see

Figure III-11C+D), which were not observed in mixtures with a low concentration of β-LG

(see Figure III-11A+B). In all other emulsion samples, also for β-LGhigh no aggregates were

found (results not shwon). The presence of aggregates decreases kinetic stability because

velocity of creaming increases with higher oil droplet size as stated by Stokes’ law. In a

recent study on interactions of QS and β-LG and the impact on emulsion stability no ag-

gregation was reported (Piotrowski et al. 2012). However, it has to be kept in mind that in

the previously mentioned study the emulsion stability was analyzed only up to 2 hours and

with sufficiently smaller β-LG-concentrations.

Figure III-11 Microscopic images of emulsions after 7 days of storage prepared with 0.005 % QS

and A) 0.005 % β-LG, B) 0.01 % β-LG, C) 0.05 % β-LG and D) 0.1 % β-LG the

black bar on the lower right represents 25 µm

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Different mechanisms may be responsible for aggregation in QS/β-LGhigh-emulsions. In

general, the addition of a surfactant solution may increase the ionic strength by shielding

of charged groups and thus weakening of electrostatic repulsion, which leads to aggrega-

tion. β-LG-stabilized emulsions can be prone to aggregation at moderate ionic strength of

150 mM due to electrostatic screening (McClements 2004). However, in the present study

the increase in ionic strength and amount of di- or multivalent counter ions by the addition

of QS extracts is negligible. Solutions prepared with 0.005 % QS in 10 mM phosphate

buffer revealed the same conductivity as pure 10 mM phosphate buffer. As indicated by a

low conductivity value for the QS-solution, electrostatic screening as a cause for aggrega-

tion can be excluded since ions were removed to a great extent during solid phase

extraction. A second mechanism is aggregation due to depletion flocculation. Depletion

flocculation describes a reversible phenomenon caused by excess protein in the aqueous

phase. However, depletion flocculation at high protein concentration is considered very

uncommon for β-LG (Dickinson 2010).

A third possible mechanism includes local electrostatic attraction. At neutral pH, QS

and β-LG are both negatively charged similar to a system containing an anionic surfactant

and β-LG. As it was emphasized before, QS has only a weak negative functional group

(-COOH) and additional anionic residues in the QS extract lead to the highly negative ζ-

potential. Interactions between two negatively charged surfactants/proteins can still occur,

although interactions between counter charged surfactants/proteins are much more com-

mon (Hansted et al. 2011). β-LG has an overall negative charge at pH 7, but individual

parts of the molecule are positively charged (Majhi et al. 2006) and offer reactive sites for

the negative functional group of QS. Aggregation behavior between QS and the negatively

charged egg lecithin was reported before presumably through hydrophobic attractive forces

(Reichert et al. 2015).

In an additional experiment, we checked whether QS or β-LG facilitate aggregation be-

tween oil droplets. Therefore, an emulsion with a fixed concentration of 0.1 % β-LG was

prepared and QS was added after emulsification to obtain a QS-concentration of

0.005/0.01/0.05 and 0.1 %. After few days of storage, substantial creaming was observed

in all emulsions and the top layer of each emulsion sample was analyzed using light mi-

croscopy (results not shown). There were few aggregates in all β-LG-emulsions with added

QS but there was no clear increase of the amount of aggregates with increasing QS concen-

tration. It can be concluded that aggregation mainly occurs when both QS and β-LG are

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present at the interface (homogenization of mixtures rather than adding QS after emulsifi-

cation). The formation of aggregates upon addition of QS to a β-LG-emulsion may be

attributed to slow displacement and structural changes of β-LG by QS, which leads to a

mixed film. Protein displacement by surfactants is well described (Dan et al. 2012; Dan et

al. 2015) and for QS and β-LG evidence was found at the air/water-interface (Böttcher et

al. 2016).

III-6 Conclusions

The present study showed that interfacial and emulsion properties of QS may distinctively

altered by the presence of beta-lactoglobulin (β-LG). Adsorption data (surface pressure af-

ter 2 s) indicate that interfacial adsorption of QS is not influenced by the presence of β-LG.

When increasing concentration of β-LG in the binary mixture, data from the dynamic in-

terfacial tension measurements confirm that the interfacial tension and thus interfacial

composition is governed by QS. Interfacial layers of mixtures of QS and β-LG show a

different rheological behavior compared to the air/water-interface, but still exhibit high di-

lational and shear moduli at the oil/water-interface. When mixed interfacial layers of QS/β-

LG were subjected to expansion, Lissajous-plots showed non-linear behavior and from the

initial steep increase after start of expansion it can be concluded that a viscoelastic network

is formed, which counteracts area expansion. This strengthens the lateral network as indi-

cated by an increase in G*, but weakens the film stability against stress upon interfacial

expansion as it occurs in dilational rheology. Shear experiments also showed that mixed

interfacial layers of QS/β-LG had rather high viscous moduli. This strengthened the hy-

pothesis that interfacial packaging is not ideal and upon shear and dilational deformation

molecules cannot orientate as ideally, which leads to high loss of imposed energy. In emul-

sion experiments aggregation behavior was observed for mixtures of QS/β-LG at high β-

LG-concentrations, while the protein-based emulsions were stable. Aggregation therefore

may be attributed to intermolecular interactions between QS and β-LG, which lead to struc-

tural changes of β-LG. The altered structure of β-LG may interact with native β-LG, which

consequently leads to aggregation. QS/β-LG-interactions in the bulk were detected before

by fluorescence quenching. Aggregation of the emulsion droplets led to a decrease in the

kinetic stability of the emulsions.

To get a better understanding of the behavior of QS at the interface, future studies should

focus on in situ measurements like ellipsometry. With this method, the thickness of the

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interfacial layer and kinetics of adsorption can be monitored and conclusions can be drawn

on the arrangement of the molecules at the interface. To visualize interfacial structures

Brewster angle microscopy would be appropriate. Although not in focus of the present

study, it has to be kept in mind that there are noteworthy differences between interfacial

properties of various QS extracts. It would be interesting to perform a systematic study on

chemical composition of the various QS extracts and the resulting interfacial properties.

With this setup it would be possible to unravel structure-function-relationships.

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Manuscript IV

Factors affecting foamed emulsions pre-

pared with an extract from Quillaja sapo-

naria Molina: oil droplet size, pH and pres-

ence of beta-lactoglobulin

Food Biophysics, V. 12, No. 2, pp. 250–260. The final publication is available at Springer

via http://dx.doi.org/10.1016/j.cis.2017.02.008.

Authors

Sandra Böttchera*

Marina Eichhorna*

Stephan Druscha

a Technische Universität Berlin, Institute for Food Technology and Food Chemistry

Department of Food Technology and Food Material Science

Königin-Luise-Str.22, 14195 Berlin

*Co-first authorship

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IV-1 Abstract

Oil is well-known to act as antifoam and destabilize foam lamellae by bridging between

two adjacent foam bubbles. It was hypothesized that an optimal oil droplet size exists with

respect to the stability of the foamed emulsions, where oil droplets are sufficiently small to

postpone bridging and amount of free surfactant is sufficient to stabilize the oil/water-in-

terface and air/water-interface. Emulsions with 0.3 % Quillaja saponin and a median oil

drop-let size between 0.2 and 2.0 µm were prepared under varying homogenization condi-

tions and characterized in a dynamic foam analyzer. Results confirmed the above-

mentioned hypothesis. Stability of the foamed emulsions increased considerably with in-

creasing pH, which was attributed to higher electrostatic repulsion between oil droplets and

the effect on the balance between disjoining pressure and capillary pressure. Stability of

foamed emulsions can be further increased when emulsifiers are added sequentially. The

emulsion may solely be stabilized by β-LG, when QS is added after emulsification stability

of the foamed emulsion is distinctly higher compared to systems with simultaneous addi-

tion of QS and β-LG. Future studies should deepen our understanding of these complex

dispersed systems by investigating other proteins and food constituents.

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IV-2 Introduction

Saponins are phytochemicals, which are widely spread in various plant species (Dinda et

al. 2010; Hänsel and Sticher 2010; Negi et al. 2013; Vincken et al. 2007). The molecular

structure of saponins is characterized by a hydrophobic aglycone and hydrophilic sugar

residues resulting in an amphiphilic character of the molecule. Botanical sources for sapo-

nins include different vegetables like asparagus or spinach, but the majority of studies

focused on extracts of the Chilean soap bark tree Quillaja saponaria Molina (QS) (Güçlü-

Üstündağ and Mazza 2007). In the past five years numerous studies investigated the inter-

facial properties of Quillaja saponins at the air/water-interface (Stanimirova et al. 2011;

Wojciechowski 2013; Golemanov et al. 2013), at the oil/water-interface (Wojciechowski

2013; Golemanov et al. 2014) as well as their use in dispersed systems like emulsions (Yang

et al. 2013; Yang and McClements 2013; Bai et al. 2016) and foams (Böttcher and Drusch

2016). Quillaja saponins have a molecular weight of about 2 kDa and are therefore larger

than common low molecular weight surfactants (<0.5 kDa) but distinctively smaller than

proteins like β-lactoglobulin (18.4 kDa).

Adsorption of low molecular weight surfactants is diffusion-limited, whereas protein

adsorption is distinctively slowed by an additional barrier resulting from steric hindrances

(Wilde et al. 2004; Wierenga and Gruppen 2010). As a consequence of its structure and

molecular weight QS has unique interfacial properties and its adsorption was described as

mixed diffusion barrier controlled (Wojciechowski et al. 2011). This implies that adsorp-

tion in comparison to low molecular weight surfactants is rather slow, but faster compared

to most proteins. At the air/water-interface high values for complex shear and dilational

moduli (with high elastic moduli) were reported, which were explained by the formation of

hydrogen bonds between neighboring QS molecules (Stanimirova et al. 2011). The for-

mation of a firm viscoelastic network is very unusual for such a small molecule, but is much

more common for proteins (Mackie et al. 2000; Petkov et al. 2000; Fainerman et al. 2010).

Due to the viscoelastic interfacial QS-film, foam lamellae are sufficiently stabilized and

counteract rapid foam collapse. In contrast, interfacial layers of QS at the oil/water-interface

exhibited low complex dilational (15 mN/m) and shear moduli (7 mN/m) with high viscous

proportion as indicated by a phase angle of around 45° for low concentrations of QS (Böt-

tcher et al. 2017). Golemanov et al. (2014) suggested that penetration of oil inbetween QS

molecules reduces viscoelasticity of the interfacial film.

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Food products often represent dispersed systems with multiple phases (oil, water and air

bubbles) and thus differ in complexity from previously examined model systems. When

discussing a system with dispersed air and oil droplets in a continuous liquid phase, differ-

ent terms were used by various research groups. A dispersion of air bubbles in a continuous

liquid phase with a relatively small gas volume fraction may be described as ‘aerated’

(Langevin 2008) or ‘bubbly’ (Kichatov et al. 2016). In many publications ‘aerated emul-

sion’ is associated with systems similar to whipped cream, in which air bubbles are

stabilized by shear-induced fat bridges (Bee et al. 1986; Leser and Michel 1999; Arboleya

et al. 2009; Kim et al. 2013). The term ‘three phase foam’ may be used when the influence

of oil droplets on stability of thin liquid films is determined (Lee et al. 2012) but was also

used to describe the effect of solid particles in a foam (Mbama et al. 1998). The readers

attention is drawn to the term ‘oil-based foam’. This term describes an aerated mixture of

oil and surfactant (non-aqueous mixtures) and is usually used in the literature on petroleum

(Sherif et al. 2015). Brun et al. (2015) used the term ‘air-in-oil-in-water emulsions’ to de-

scribe an oil-based foam (dispersed air bubble in oil), which was afterwards dispersed in an

aqueous solution. In the present study the term ‘foamed emulsion’ is used to describe an

emulsion, in which a gaseous phase is dispersed (Kichatov et al. 2016).

The presence of oil significantly affects foamability and foam stability of an aqueous

system. Denkov (2004) reviewed two destabilization mechanisms of foams by oil droplets

(‘antifoams’): bridging-stretching und bridging-dewetting. Both mechanisms require an oil

bridge formed by an oil droplet between two adjacent foam bubbles. In the former mecha-

nism the oil bridge is stretched due differences in the Laplace pressure in the aqueous phase

and drainage, which leads to the formation of a biconcave oil bridge. Upon stretching of

the film the oil bridge thins and film rupture may occur. Thinning of the oil bridge cannot

be slowed down by usual processes of film stabilization like the Gibbs-Marangoni mecha-

nism. When considering the bridging-dewetting mechanism, the bridging oil droplet is

dewetted by the surrounding aqueous phase due to the hydrophobicity of the oil. The joint

interface between aqueous solution and oil droplets decreases due to the dewetting, which

subsequently leads to film rupture. In general, larger oil droplets tend to bridge foam lamel-

lae faster than smaller oil droplets, because bridging of oil droplets happens when lamella

thickness is similar to the size of the oil droplet.

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Figure IV-1 Schematic illustration of the relationship between the stability of foamed emulsions

and their oil droplet size. Please refer to the text for appropriate explanation of the

different ranges.

In addition, to the oil drop size the content of the emulsifier is another factor, which may

affect the stability of the foamed emulsion. At a fixed content of the emulsifier, a higher

proportion is required for the stabilization of the oil/water-interface at a small oil droplet

size compared to a large oil droplet size. The interfacial area is distinctively larger in emul-

sions with small oil droplet size because the area-to-volume-ratio is higher. As a

consequence, free surfactant content may be insufficient to stabilize the air/water-interface

of air bubbles, which reduces stability of the foamed emulsion. It must therefore be hypoth-

esized that there is an optimal oil droplet size of emulsions with maximum stability of a

foamed emulsion (see range 2 in Figure IV-1). Below the optimal oil droplet size, in range

1 in Figure IV-1, the concentration of non-adsorbed emulsifier is too small to stabilize the

air/water-interface in addition to the oil/water-interface. Above the optimal oil droplet size

(see range 3 in Figure IV-1) stability of the foamed emulsions is reduced by bridging of the

oil droplets, which leads to rupture of the foam lamella.

As reported in a previous studies the stability of QS-based foams and emulsions is distinctly

affected by changes in pH (Böttcher and Drusch 2016; Yang et al. 2013), thus representing

a third factor affecting foam properties. Quillaja saponins possess a free carboxylic group

and since it’s an aqueous plant-derived extract contains other constituents, which may carry

ionic residues. At neutral pH anionic residues and the carboxylic group of QS are negatively

charged, which leads to electrostatic repulsion in thin liquid films and between oil droplets.

oil droplet size [µm]

Stability of foamed emulsion

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It may therefore be hypothesized that stability of foamed emulsions at increased at a higher

pH.

Due to the fact that QS is approved for some food products in the EU and US, various

studies investigated the interactions with typical food ingredients e.g. β-lactoglobulin in

model systems (Kezwon and Wojciechowski 2014; Böttcher et al. 2016). In previous pub-

lications mixtures of QS and β-LG showed synergistic and antagonistic effects with respect

to the stability of dispersed systems. In general, QS and β-LG form complexes in bulk as

demonstrated by fluorescence experiments (Böttcher et al. 2016). In foam experiments it

was shown that foaming speed, foam stability and liquid content of the foam increase when

using mixtures of QS and β-LG (Böttcher et al. 2016). The observed synergistic behavior

in foams at low concentrations of QS and β-LG was explained by the formation of a joint

interfacial network. Interfacial shear and dilational rheology supported this as indicated by

an increase of the complex shear and dilational moduli. However, for emulsions an antag-

onistic effect on the kinetic stability due to extensive oil droplet aggregation was reported

for binary mixtures of QS and β-LG (Böttcher et al. 2017). It was hypothesized that QS

may prompt structural changes or partial displacement of β-LG which caused aggregation.

The situation in foamed emulsions is different, since two different interfaces exist, which

are formed in two subsequent steps of the process. As a consequence, physical stability will

depend on the fact, whether proteins and saponins are already present during emulsion prep-

aration or not. Depending on desorption and re-adsorption phenomena as well as the

specific behavior of an emulsifier at these interfaces, it may be necessary to provide a spe-

cific emulsifier for each step of the formation of this more complex dispersed system.

Aim of the present study therefore is to identify key factors increasing the stability of

foamed emulsions prepared with Quillaja saponins. In a first step the influence of oil droplet

size (d50 0.2 to 2 µm) and amount of free emulsifier on the stability of foamed emulsions is

investigated. Emulsions are characterized with respect to oil droplet size distribution, ζ-

potential and interfacial tension. Stability of foamed emulsions is evaluated with an auto-

matic foam device. In a second step, the impact of pH within a range of pH 2 to 8 on stability

of foamed emulsions is determined. Finally, the effect of the presence of β-lactoglobulin in

a QS-based system is investigated by performing experiments with sequential addition of

both constituents.

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IV-3 Material and methods

A crude saponin extract from Quillaja saponaria Molina (QS) with a purity of 15.5 % was

kindly provided by Ingredion Germany GmbH (Hamburg, Germany). Beta-Lactoglobulin

(β-LG) with a purity of 85 % was purchased from Davisco Foods International, Inc. (Ge-

neva, Switzerland). Medium-chain triglyceride oil (MCT-oil) WITARIX® MCT 60/40

containing a mixture of C:8 and C:10 triglycerides, was chosen as oil phase and was pur-

chased from CREMER OLEO GmbH & Co. KG (Hamburg, Germany). Surface-active

residues present in the MCT-oil were removed through adsorption onto magnesium silicate

(Florisil®, Sigma-Aldrich GmbH, Seelze, Germany). For coloring the oil phase 0.017 %

Oil Red O was used. All solutions of QS and β-LG were prepared in potassium phosphate

buffer (10 mM, pH 7). For the experiment with variation of the pH, emulsions were ho-

mogenized using distilled water as aqueous phase and pH was adjusted afterwards within a

range of 2 to 8 using hydrochloric acid and sodium hydroxide with a concentration of 0.01

IV-3.1 Preparation of emulsions

Emulsions (o/w) were produced in two steps: first, a coarse emulsion was prepared using a

rotor-stator system (Ultra-Turrax T25 basic, IKA -Werke GmbH & CO. KG, Staufen, Ger-

many) with an adjusted speed of 13,500 rpm, which was applied for 30 s. Aqueous solutions

containing QS, β-LG and mixtures of both were emulsified with 5 % of purified and dye-

containing MCT-oil. Afterwards, the coarse emulsion was homogenized to obtain smaller

oil droplets by using a high-pressure homogenizer (Panda 2K, GEA Niro Soavi Deutsch-

land, Lübeck, Germany). In the first experiment on the impact of oil droplet size, pressure

regime ranged between 50-600 bar and number of passes was varied between one and four

in order to identify suitable conditions to produce emulsions with a median of the oil droplet

size distribution between 0.2 and 2 µm (Table IV-1). In all other experiments emulsions

with a d50 of 0.5 µm were prepared applying 200 bar and three passes.

In all emulsion experiments, QS concentration was fixed at 0.3 %. In emulsions containing

β-LG, the β-LG concentration was varied between 0.1, 0.2 and 0.3 %. Throughout the

whole manuscript Mixpre describes the process, when QS and β-LG are co-dissolved prior

to emulsification and Mixpost indicates that a β-LG emulsion was produced first and QS is

added after emulsification.

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Table IV-1 d50 of 0.3 % QS-emulsions (5 % MCT-oil) in relation to number of passes and ho-

mogenization pressure; bold numbers refer to chosen parameters for experiment on

foaming of emulsions

pressure [bar]

1 pass

2 passes

3 passes

4 passes

3.7

2.2

2.1

1.8

100

2.3

1.2

1.0

0.8

200

1.4

0.6

0.5

0.4

300

0.8

0.4

0.3

400

0.6

0.3

500

0.4

0.3

0.2

600

0.4

0.2

IV-3.2 Oil droplet size distribution, ζ-potential and interfacial tension of the emulsions

A static light scattering device (Horiba LA-950, Retsch Technology GmbH, Haan, Ger-

many) was used to analyze the oil droplet size distribution of emulsions. Based on the

scattering patterns the volume distribution of the oil droplets is calculated and reported.

Emulsions need to be diluted to obtain an optimal scattering pattern. Circulation and stirring

speed were set to 8 and 3, respectively. For all calculations, a refractive index of 1.45 was

used. Box plots comprising different quantiles (d10, d25, d50, d75 and d90) are used to describe

differences in the oil droplet size distribution.

The ζ-potential of dispersed oil droplets was determined via the electrophoretic mobility

using a Nano Zetasizer ZS from Malvern Instruments GmbH (Herrenberg, Germany). Ex-

periments were carried out in clear disposable cells (DTS 1060 C, Malvern Instruments

GmbH) and emulsions were diluted 1:10 using 10 mM phosphate buffer prior to measure-

ment.

Drop shape analysis is a convenient possibility to determine dynamic interfacial tension. In

this study contact angle meter in pendant drop mode was used (OCA-20, DataPhysics In-

struments GmbH, Filderstadt, Germany). A droplet of emulsion (15 µL± 0.3) was

automatically generated through a needle. Droplets were measured at 22 °C in a closed

cuvette filled with a small amount of water to prevent extensive evaporation. The device

calculates interfacial tension in real-time by fitting the Young-LaPlace-equation to the drop

shape every 3 s for 20 mins (Najmabadi et al. 2013).

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IV-3.3 Preparation and characterization of foamed emulsions

Dynamic foam properties (foaming and foam stability) of the emulsions were analyzed us-

ing a DFA 100 (Krüss GmbH, Hamburg, Germany) as described in Böttcher and Drusch

(2016). For each measurement, an aliquot of 50 ml of emulsions was foamed to a maximum

height of 180 mm by purging pressurized air (0.2 L/min) through a porous glass frit (FL

4504, Porosity 4, 10-16 µm, DURAN®) at the bottom of a cylinder. In this experimental

setup foam and liquid height are typically differentiated by differences by their transmissi-

bility. Due to opacity of the emulsions, discrimination between foam and liquid height was

not possible. Therefore, stability was evaluated by comparing the total height, which is the

sum of foam height and liquid height. All experiments were carried out in darkness over a

period of 3600 s. Transmission was recorded every 0.5 s for layers of a height of 0.1 mm

covering the whole foam column to generate the foam profile. The foam profile is thus

composed of grey pixels and brightness values vary between 0 and 255, which refers to

black and white, respectively.

Based on this foam profile the median of the brightness distribution (BDm) was derived at

3600 s (for detailed explanation refer to Böttcher and Drusch (2016)). The BDm may be

interpreted as an indicator for density of the foamed emulsion. The amount of transmitted

light is reduced at high foam density, which results in a low BDm. In contrast, a high value

for BDm indicates a porous foam.

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IV-4 Results and discussion

IV-4.1 Impact of homogenization parameters on oil droplet size distribution of emul-

sions

With increasing homogenization pressure and number of passes, the median (d50) of the oil

droplet size distribution decreased and width of the oil droplet size distribution decreased.

Figure IV-2 shows the dependence of the oil droplet size on the number of passes (see Panel

A) and homogenization pressure (see Panel B). At a pressure of 200 bar after one pass the

width of the oil droplet size distribution (d10 to d90) amounted to 1.6 µm. With increasing

number of passes the width of the oil droplet size distribution steadily decreased to 0.7 µm

at four passes. A similar trend was observed when comparing the oil droplet size distribu-

tions at 2 passes at varying pressure. The width of the oil droplet size distribution decreased

from 2.4 µm at 50 bar to 0.3 µm at 600 bar. As expected, median of the oil droplet size

distribution was highest at 50 bar (2.2 µm) and decreased with increasing pressure. Above

300 bar the median of all samples were in a similar range between 0.2 to 0.4 µm.

Figure IV-2 Influence of (A) number of on the oil droplet size distribution of 0.3 % QS-emulsion

(200 bar, 5 % MCT-oil) with upper whisker representing d90, upper box end d75,

dash in the box d50, lower box end d25 and lower whisker d10 and (B) influence of

homogenization pressure on the oil droplet size distribution of 0.3 % QS-emulsion

(2 passes, 5 % MCT-oil)

50 100 200 300 400 500 600

oil droplet size [µm]

pressure [bar]

1234

oil droplet size [µm]

number of passses [-]

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The impact of the number of passes and homogenization pressure on oil droplet size is well

known and has been described before for QS-emulsions by some authors. In the present

study QS content and oil content differed from the experimental setup described in the lit-

erature. Therefore, it was important to identify homogenization parameters, which result in

specific values for the d50 between 0.2 and 2 µm. At increasing homogenization pressure

and number of passes stress increases and oil droplet size is reduced. Reduction in oil drop-

let size goes along with an increase of the area-to-volume-ratio and an increase of the total

interfacial area. A linear relationship between the logarithm of the d50 of the volume distri-

bution and the logarithm of the homogenization pressure was observed (see Figure IV-3)

and is in agreement with the literature (Yang et al. 2013). It is generally known that an

inverse linear relationship between the logarithm of the diameter of the oil droplets and the

logarithm of the energy density exists as long as sufficient emulsifier is present in a system.

The latter results from the general definition of the interfacial tension as amount of work

required to create a specific new interfacial area.

Figure IV-3 Double logarithmic plot of the relationship between d50 and homogenization pressure

of a 0.3% QS-emulsion (5 % MCT-oil) for 1 pass (■), 2 (▲), 3 (●) and 4 passes (♦)

-0,8

-0,4

0,0

0,4

0,8

1,5 2,0 2,5 3,0

log (d50 [µm])

log (pressure [bar])

0.8

0.4

0.0

-0.4

-0.8

1.5

2.0

2.5

3.0

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IV-4.2 Emulsion properties and stability of foamed QS-emulsions affected by the oil

droplet size

The interfacial tension of an emulsion droplet against air provides information on the

amount of free surfactant, which is not adsorbed to the oil/water-interface and is available

to stabilize the air/water-interface. As outlined above with decreasing oil droplet size of an

emulsion the total interfacial area increases and vice versa. Therefore, interfacial tension of

the emulsions decreased with increasing d50 from 56 to 49 mN/m between 0.2 and 0.4 µm,

respectively (see Figure IV-4A). Above a d50 of 0.5 µm interfacial tension did not change

upon further increase of d50. High values of the interfacial tension indicate a low concen-

tration of QS at the air/water-interface and thus a low content of free emulsifier.

The amount of free QS affects the foaming speed of an emulsion. Free QS adsorbs at the

air/water-interface to stabilize newly formed air bubbles and prevents rapid coalescence.

As a consequence, foaming speed of the emulsions increased to a maximum of 2.85 mm/s

at a d50 of 0.6µm (Figure IV-4A). Thereafter foaming speed remained constant and slightly

decreased above a d50 of 1 µm. Low foaming speed originates from low amount of free

surfactant as it is indicated by high values for the interfacial tension.

Before discussing the impact of the oil droplet size on foam stability, general mechanisms

responsible for changes in dispersity of foams need to be summarized. Foams and their

stability have been a topic of extensive research (Langevin 2008; Aveyard et al. 1999;

Stubenrauch and von Klitzing 2003; Fauser and von Klitzing 2014; Schramm 2005). Foams

are thermodynamically instable and may be classified based on the relative content of the

dispersed phase. Foams with a low content of dispersed phase may be referred to as wet

foam (‘kugelschaum’) and foams with high content as dry foams (polyhedral). In polyhe-

dral foams two adjacent air bubbles are separated by a thin liquid film (foam lamella). Three

lamellae are joined by a Plateau border and four Plateau borders will form a so-called node

in a three dimensional arrangement.

Different destabilizing mechanisms resulting in a change of dispersity may occur in foams:

1) evaporation of gas, 2) disproportionation of bubbles, 3) drainage, followed by 4) thin

liquid film rupture and coalescence of adjacent bubbles (Aveyard et al. 1999; Langevin

2008). We expect that disproportionation, drainage and coalescence have the highest impact

on foam stability in the present study. Evaporation of gas plays a minor role in this study

because the surface of the foam in the column of the foam apparatus only amounts to

12.5 cm². Disproportionation originates from differences in the Laplace pressure in bubbles

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in polydisperse foams. Small bubbles have a high Laplace pressure, which increases solu-

bility of gas in the adjacent liquid phase. In the vicinity of a large bubble (with a low Laplace

pressure) solubility of the gas in the continuous phase is reduced. The excess gas is released

into the large bubble until equilibrium is achieved. Disproportionation results in shrinkage

of small bubbles in favor of larger bubbles and is the dominating mechanism in foams.

Drainage (process 3) through the Plateau borders is caused by gravitational forces and dif-

ferences in capillary pressure between the lamellae and the plateau border region pull out

liquid from the lamellae. Immediately after their formation, liquid films between bubbles

are thick but draining leads to the development of thin liquid films. In polyhedral foams,

adjacent foam bubbles form a planar thin liquid film in the center, but in the Plateau border

region foam bubbles are curved. Curvature of the bubbles leads to a reduction in pressure

in the Plateau borders (as described by Young-Laplace equation). The pressure difference

between the Plateau border regions and the center of the thin liquid film leads to drainage

of the continuous phase, which reduces thin liquid film thickness and increase tendency of

film rupture due to holes in the interfacial film (process 4). The Gibbs Marangoni mecha-

nism may counteract thinning by a countercurrent liquid flow due to interfacial tension

gradients. Disjoining forces are contradicting the thinning process of thin liquid films at

small thickness (10-100 nm) of thin liquid films (Aveyard et al. 1999; Langevin 2008). As

described in the introduction of the manuscript oil droplets may decrease foam stability by

bridging of air bubbles followed by coalescence due to stretching and film rupture and or

dewetting.

In the present study decay of the foamed emulsions was not necessarily indicated by a de-

crease in the height of the foam column. Also density of the foamed emulsions distinctly

changed over the course of the measurement. Therefore, the brightness of the foam profiles

was analyzed and the median of the brightness distribution (BDm) was chosen. Generally

spoken, a low value for the BDm indicates a denser foam. The BDm after 1 h showed a

minimum value of approximately 100 for foamed emulsions with an oil droplet size be-

tween 0.6 to 1 µm (see Figure IV-4B). At a d50 above 0.5 µm the BDm values were

approximately 115. At a d50 below 0.5 µm foamed emulsions were unstable and rapidly

collapsed after the end of foaming. Therefore, no values for BDm are reported.

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Figure IV-4 Emulsion properties with respect to (A) interfacial tension σ (□) and foaming speed v

(◇) and (B) properties of the foamed emulsions described by the median of the

brightness distribution BDm after 1 h measurement (△) in relation to the median

(d50) of 0.3 % QS-emulsions with range 1, 2 and 3. Lines are only guide to the eye.

Please refer to the text for appropriate explanation of the different ranges.

The results of the interfacial tension, foaming speed and stability of the foamed emulsions

confirmed the hypothesis that there is an optimal oil droplet size at which stability of the

foamed emulsions is maximized. Thus, three ranges of the oil droplet size with differing

stability may be described (see Figure IV-4, Figure IV-5).

012

v [mm/s]

σ[mN/m]

d50 [µm]

100

110

120

012

BDm[-]

d50 [µm]

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Figure IV-5 Foam profiles of foamed emulsions of 0.3 % QS with d50 of

A) 0.2 µm, 600 bar, 3 passes; B) 0.3 µm, 500 bar, 2 passes; C) 0.4 µm, 300 bar, 2

passes; D) 0.5 µm, 100 bar, 3 passes; E) 0.6 µm, 200 bar, 2 passes; F) 0.8 µm,

100 bar, 4 passes; G) 1 µm, 100 bar, 3 passes; H) 1.2 µm, 100 bar, 2 passes; I)

2.1 µm, 50 bar, 3 passes

In the first range up to a d50 of 0.4 µm a large proportion of QS is adsorbed to the oil/water-

interface. The amount of free surfactant is too low to stabilize the air/water- after stabilizing

the oil/water-interface, as indicated by high interfacial tension values. Rapid bubble coales-

cence during foaming and after the end of foaming occurred and hence foaming speed and

Time

Total height

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stability of the foamed emulsions were low. Characteristic properties of emulsions belong-

ing to the first range are a high specific interfacial area of the oil droplets (o/w-interface).

E.g. at a d50 of 0.2 µm the interfacial area amounts to 326,000 cm²/cm³ (see Figure IV-6).

In the second range sufficient amount of QS-molecules is available to stabilize the oil/wa-

ter-interface as well as the air/water-interface during foaming. Bridging of foam lamellae

by oil droplets still occurs but is low due the relatively small size of the oil droplets (Denkov

2004). Emulsions with a d50 between 0.6 to 1 µm represent this range and may be consid-

ered as the optimum range of oil droplet size with respect to stability of foamed emulsions

in the present system at a concentration of 0.3 % QS and 5 % MCT-oil.

In the third, range covers emulsions with a d50 above 1 µm. In this range enough free QS-

molecules are present to stabilize both interfaces: oil/water and air/water. The specific in-

terfacial area of an emulsion with a d50 of 2.1 µm (31,000 cm²/cm³, see Figure IV-6) in the

present study was 10-times smaller in comparison to an emulsion with a d50 of 0.2 µm.

Foaming speed and interfacial tension are similar compared to the second range but stability

of the foamed emulsion was lower. With increasing measurement time the formation of

light areas along the brightness profile of the foam column were observed, which indicate

foam decay in this area.

Figure IV-6 Specific interfacial a rea Aspec in relation to d50 of 0.3% QS-emulsions

As discussed by Karakashev and Grozdanova (2012) and Denkov (2004) the antifoam prop-

erties of oil may be classified based on the speed of foam destruction. Oil may act as slow

or fast antifoam, which means that foams are either destroyed in seconds or minutes to

hours by the presence of the oil, respectively. Fast antifoams have a low entry barrier, which

may be determined with the ﬁlm trapping technique (FTT) and destroy thin liquid films in

100.000

200.000

300.000

400.000

0123

Aspec [cm²/cm³]

d50 [µm]

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an early stadium. The destruction of the thin liquid films occurs only seconds after thin

liquid film formation thereby preventing foam generation. However, slow antifoams have

a high entry barrier and are trapped in the Plateau boarders, because they are not able to

enter the foam film. Drainage of the liquid phase increases compression of slow antifoam

in the Plateau borders consequently leading to the rupture of Plateau borders. In the present

system, it is assumed that dispersed MCT-oil acts as a slow antifoam, because foam de-

struction is relatively slow.

All trials described in the following subchapter were conducted with emulsions, which had

a d50 of 0.5 µm. The oil droplet size is lower in comparison to the optimal oil droplet size

(range 2), which results in reduced stability of the foamed emulsions (see Figure IV-5D+H).

This oil droplet size was chosen on purpose to enable the determination of factors that pos-

itively influence stability of foamed emulsions.

IV-4.3 Influence of pH on ζ-potential and stability of foamed QS-emulsions

A pH-series of QS-emulsions with pH ranging from 2 to 8 were prepared and foamed. In

Figure IV-7 foam profiles are displayed, which show the total height in relation to the meas-

uring time of QS-emulsions with a pH 3, 5 and 7.

Figure IV-7 Foam profile showing total height in relation to measuring time for foamed emul-

sions (5 % MCT-oil, d50=0.5 µm) prepared at A) pH 3 B) pH 5 and C) pH 7. A was

analyzed for 1800 s, B and C for 3600 s

At low pH of 2 and 3 foamed emulsions rapidly collapsed after the end of foaming within

less than 10 minutes. At pH 5, foamed emulsions were distinctly more stable and did not

show any decay, but with increasing time, light regions developed in the foam profile.

Foamed QS-emulsions with a pH above 5 showed no decay and almost no light regions

during the course of the measurement. With increasing pH, ζ-potential decreased from -

11 mV at pH 2 to -58 mV at pH 8 (see Figure IV-8).

Time

Total height

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Figure IV-8 ζ-potential in relation to pH of 0.3 % QS-emulsions (5 % MCT-oil, d50=0.5 µm)

Increased aggregation tendency of oil droplets in QS-emulsions at pH 2 was shown before

(Yang et al. 2013) and was observed as well in the present study. In Figure IV-9 rapid

aggregation of a foamed QS-emulsion at pH 2 at the end of the foam measurement is shown.

With a decrease of the absolute value of the ζ-potential, electrostatic repulsion between oil

droplets is reduced, which facilitates aggregation of oil droplets. In non-foamed emulsions,

a ζ-potential with an absolute value above 30 mV is considered to slow down aggregation

(Heurtault et al. 2003). Aggregates of oil droplets may bridge foam lamellae because of the

increased diameter, which leads to film rupture and rapid foam decay. A highly negative ζ-

potential of QS-emulsions at increasing pH may be attributed to the carboxylic group of QS

and other constituents with ionic residues in the crude extract and enforces repulsion be-

tween oil droplets.

As it was discussed before, that in the present study, MCT-oil is considered to act as a slow

antifoam and oil droplets at low pH accumulate in the Plateau borders. The electrostatic

repulsion of oil droplets opposes compression of oil droplets in the Plateau borders due to

drainage, which decelerates rupture.

However, the increasing foam stability with increasing pH cannot fully be explained by ζ-

potential. At pH 5 ζ-potential amounted to -48 mV, which indicates increased electrostatic

repulsion between oil droplets, but still, lighter regions in the foam profile appeared. Foams

at pH 5 and 6 were less stable compared to pH 7 and 8, but ζ-potential between pH 5 to 8

did not distinctively change.

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ζ[mV]

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Figure IV-9 Foamed emulsion of 0.3 % QS (5 % MCT-Oil, d50=0.5 µm) after 1800 s with macro-

scopic aggregation of oil droplets

An additional factor explaining the increase in foam stability with increasing pH is a dif-

ference in disjoining pressure, and thus the behavior of the thin liquid films. Disjoining

pressure Π is described as a function of film thickness ht, and depends on the interaction

forces, e.g. long-range repulsive electrostatic (Πel), short-range attractive van der Waals

(Πvw) and short-range repulsive steric (Πsteric). The former two forces are described by the

DLVO-theory developed from Derjaguin, Landau, Verwey and Overbeeek. The disjoining

pressure depends on the thickness of the thin liquid film and the relationship between the

two is expressed in disjoining pressure isotherms. In some thin liquid films metastable states

like common black films (10-100 nm) and Newton black films (~1 nm) may arise at a spe-

cific film thickness. The former is more thick and stabilized by electrostatic repulsion and

the latter is sterically (entropically) stabilized. At these metastable states disjoining pressure

is equal to capillary pressure, which prevents further film thinning. (Stubenrauch and von

Klitzing 2003; Fauser and von Klitzing 2014; Schramm 2005)

It may be hypothesized that pH adjustment influences electrostatic properties of surface-

active residues from the QS-extract, which are adsorbed at the air/water-interface and con-

tribute together with non-electrostatic interactions to stabilization. Furthermore difference

in the physicochemical properties of the dispersed phase (air vs. MCT oil) may result in an

inhomogeneous distribution of anionic constituents at the air/water and oil/water-interface.

Finally it is well described in the literature that disjoining pressure is affected by ionic

strength of the medium, which is slightly altered because of pH adjustment. All these factors

may affect the balance between disjoining pressure and capillary pressure in thin films and

contribute to increased foam stability at higher pH although ζ-potential does not change at

pH higher than 5.

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IV-4.4 Emulsification of a binary mix of QS and β-lactoglobulin and the influence on

the stability of foamed emulsions

As outlined in the introduction, food products often represent complex dispersed systems

with a multitude of different constituents. Proteins may affect interfacial properties of dis-

persed systems by adsorbing onto aqueous interfaces and their interaction with other surface

active constituents in the bulk and at the interface. Therefore, molecular interactions and

the presence of more than one surface active constituent during processing need to be con-

sidered. In this chapter Mixpre refers to samples in which QS and β-LG were mixed prior to

emulsification and Mixpost specifies β-LG emulsions to which QS was added after emulsi-

fication.

Distribution of QS and β-lactoglobulin at the air/water- and oil/water-inter-

face

ζ-potential of QS-, β-LG-, Mixpre- and Mixpost-emulsions was measured to characterize elec-

trostatic at the shear plane close to the interface of the dispersed oil droplets (oil/water-

interface). ζ-potential of Mixpost was distinctly different from Mixpre, however ζ-potential

was in both cases independent from the β-LG concentration (see Figure IV-10). For Mixpre-

emulsions a ζ-potential of -62 to -56 mV was determined at β-LG concentrations of 0.1 and

0.3 %, respectively. The absolute values of the ζ-potential of Mixpre-emulsions were higher

than the ζ-potential of emulsions solely prepared with QS or β-LG. In contrast, with ap-

proximately 40 mV, the absolute value of the ζ-potential of Mixpost-emulsions was

considerably lower and similar to emulsions solely prepared with β-LG.

In addition to the ζ-potential the interfacial tension of QS-, β-LG-, Mixpre- and Mixpost-

emulsions was used to describe interfacial characteristics at the air/water-interface. It

should be noted that interfacial tension values were compared after 150 s because shortly

after this time period in Mixpost-emulsions the droplet detached from the tip of the needle

of the device (results not shown).

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Figure IV-10 ζ-potential of emulsions (0.3 % QS, 5 % MCT-oil, d50=0.5 µm) in relation to β-LG-

concentration for QS (○), β-LG (◇), Mixpre (△) and Mixpost (□)

From the results of the ζ-potential it can be hypothesized, see illustration in Figure IV-11A

on the left, that in Mixpre-emulsions a mixed film containing QS and β-LG is formed at the

oil/water-interface, which is dominated by QS. It was discussed in previous publications

(Böttcher et al. 2016, Böttcher et al. 2017) that QS is a small molecule, which adsorption

at aqueous interfaces is slower than for common low molecular weight surfactants but faster

in comparison to β-LG. However it is very likely at both interfaces (air/water- and oil/water)

a mixed film of QS and β-LG is formed. The high absolute values of the ζ-potential may be

explained by interactions between QS and β-LG. In Mixpre a co-adsorption of QS and β-

LG may take place (due to interactions of both), which increases overall surface coverage

at the oil/water-interface, which increased the absolute value of the ζ-potential.

In contrast in Mixpost-emulsions oil droplets were predominantly covered by β-LG, which

can be deduced from the similarity of the ζ-potential to β-LG (see Figure IV-11C). That

means when QS is added after the emulsification QS is available to primarily stabilize the

air/water-interface. Once the oil/water-interface is covered by β-LG co-adsorption with QS

cannot take place, which results in ζ-potential similar to β-LG. It is yet unclear whether

sequentially added QS may fully desorb β-LG from the oil/water-interface. The desorption

of β-LG by QS was shown in experiments at the air/water-interface. But it has to be noted

that β-LG considerably unfolds at the oil/water-interface (Zare et al. 2016, Zhai et al. 2017)

thus it may be hypothesized that desorption is less likely at the oil/water-interface.

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Figure IV-11 Schematic illustration of location of QS (green) and β-LG (violet) in emulsions

(A+C) and foamed emulsions (B+D) whereas Mixpre is illustrated in A+B and

Mixpost in C+D

Influence of distribution of QS and β-lactoglobulin on foaming and stability

of foamed emulsions

As discussed in the previous chapter, it is hypothesized that in Mixpre-emulsions a mixed

film of QS and β-LG is present at the air/water- as well as oil/water-interface. In Mixpost-

emulsions ζ-potential and interfacial tension data indicated that the oil/water-interface is

primarily stabilized by β-LG and that sequentially added QS is available to stabilize the

air/water-interface.

Foam experiments were performed to determine the impact of the inhomogeneous distribu-

tion of QS and β-LG at the air/water- and oil/water-interface on foaming properties and

foam stability. Figure IV-12 presents results of the foaming speed of QS-, β-LG-, Mixpre-

and Mixpost-emulsions. The highest values for the foaming speed with approximately 3

air

oil

water

Emulsion

Foamed emulsion

foaming

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mm/s were obtained for β-LG-emulsions (with a concentration above 0.2 %) and all Mixpre-

emulsions. In contrast with 2.8 mm/s, foaming speed of QS was slightly lower. Lowest

foaming speed was measured for Mixpost samples, ranging from 2.5 to 2.94 mm/s with in-

creasing β-LG concentration.

Figure IV-12 Foaming speed of emulsions (0.3 % QS, 5 % MCT-oil, d50=0.5 µm) in relation to β-

LG-concentration for QS (○), β-LG (◇), Mixpre (△) and Mixpost (□)

In Figure IV-13 foam profiles of foamed emulsions containing QS (A), β-LG (B-D), Mixpre

(E-G) and Mixpost (E-J) are displayed. Light patches appeared in the middle of the column

of the foamed QS-emulsions after half of the measurement time, but total height of the foam

column did not decrease. In contrast, total height of the foamed β-LG-emulsions rapidly

decreased after end of foaming, but decay was slowed down at higher β-LG-concentrations.

When foaming Mixpre-emulsions light regions appeared after 30 mins and with increasing

β-LG concentration the size of the light patches increased. In contrast, foam profile of

Mixpost-emulsion neither exhibited any decay in total height nor light patches during the

course of the measurement.

00,1 0,2 0,3

v [mm/s]

cBLG [%wt]

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Figure IV-13 Foam profiles showing total height in relation to measuring time for a foamed

emulsion (d50=0.5 µm) prepared with 5 % MCT-oil and (A) 0.3 % QS; (B) 0.1 % β-

LG; (C) 0.2 % β-LG;(D) 0.3 % β-LG; (E) 0.3 % QS+0.1% β-LG (Mixpre); (F) 0.3 %

QS+0.2 % β-LG (Mixpre); (G) 0.3 % QS+0.3% β-LG (Mixpre); (H) 0.3 % QS+0.1%

β-LG (Mixpost); (I) 0.3 % QS+0.2 % β-LG (Mixpost);

(J) 0.3 % QS+0.3% β-LG (Mixpost)

Time

Total height

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Summarizing results of the foam experiments it can be stated that foaming speed of Mixpre-

emulsions was faster in comparison to Mixpost but that stability of the Mixpost-emulsion was

distinctly higher. Foaming speed and stability of the foamed emulsions are tremendously

affected by the decision on the process step, during which QS is added, although the total

emulsifier concentration is similar in both experimental setups.

As discussed above in Mixpre-emulsions QS and β-LG form a mixed film at both interfaces:

air/water and oil/water (see Figure IV-11B+D). Foaming speed of Mixpre-emulsion was

higher than QS and Mixpost, which supports previously, reported synergistic effect of QS/β-

LG on foaming properties (Böttcher et al. 2016). As stated in the previous chapter, a co-

adsorption of QS and β-LG may not only occur at the oil/water-interface but also at the

air/water-interface. Thus, foaming speed increases due to higher surface coverage at the

air/water-interface. But the antagonistic effect of QS and β-LG in emulsions led to a de-

crease in stability of the foamed emulsion. Stability of foamed emulsions of Mixpre reduced

with increasing β-LG concentration as indicated by the formation of light patches in the

foam profile. This behavior is unusual because the overall concentration of surface-active

constituents increased. We therefore took microscopic images of the Mixpre-emulsions (re-

sults not shown) and distinct coalescence of oil droplets was observed. As discussed before

in this manuscript, large oil droplets lead to bridging of thin liquid films and subsequently

to film rupture.

However, it was shown that stability of the foamed emulsion can be increased by sequen-

tially adding QS to a β-LG-emulsion (Mixpost). In this experimental setup the presence of

QS at the air/water-interface and the stabilization of the oil/water-interface by β-LG led to

a considerably higher stability of the foamed emulsions (see Figure IV-13D).

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IV-5 Conclusion

The present study clearly showed that oil droplet size and pH as well as the presence of β-

LG and its use as an emulsifier for the oil/water-interface are key factors for foamability

and foam stability of foamed QS-emulsions. At a constant QS-concentration there is an

optimal range of the oil droplet size of emulsions, at which the stability of the foamed

emulsion is maximized. Above the optimal oil droplet size, emulsions are easily foamed,

but stability is limited due to fast occurrence of bridging induced by large oil droplets.

Below the optimal oil droplet size, amount of free surfactant is too low to stabilize the

air/water- and oil/water-interface, which results in low foamability and poor stability. It

was further concluded that dispersed MCT-oil in the system studied acts as a slow antifoam.

Slow antifoams may not enter the thin liquid film between two adjacent bubbles, but rather

accumulate in the Plateau border regions and induce film rupture with increasing drainage.

Stability of the foamed emulsions increased considerably with increasing pH, which was

attributed to higher electrostatic repulsion between oil droplets and the effect on the balance

between disjoining pressure and capillary pressure. The pH sensitivity originates from the

carboxylic group of QS and constituents carrying ionic groups which are present in the QS-

extract. Stability of foamed emulsions further increased when emulsifiers are added se-

quentially. The emulsion may solely be stabilized by β-LG. When QS is added after

emulsification it is available to stabilize the air/water-interface. As shown in previous pub-

lications, β-LG efficiently stabilizes an oil/water-interface and QS forms a strong

viscoelastic network to stabilize the air/water-interface. In this sequential approach the op-

timal properties of QS and β-LG are beneficially used to maximize stability.

Complex systems like foamed emulsions, which contain more than two phases, are chal-

lenging systems and various factors may influence stability. Although this study revealed

important factors that influence stability of foamed emulsions, in future studies the impact

of other (food) constituents like inorganic salts and hydrocolloids should be investigated.

Charged groups may influence electrostatic conditions and hydrocolloids increase viscosity

of the liquid phase, which may increase stability of the foamed emulsions. Their general

impact on foams can be described from the literature, but their specific contribution and

interrelations in these more complex systems need to be characterized in more detail. Future

studies should also focus on other proteins like random coil proteins with different interfa-

cial properties in comparison to globular proteins.

General discussion

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6. General discussion

This dissertation aimed to deepen understanding of the behavior of saponins at air/water-

and oil/water-interfaces by combining interfacial rheology with experiments in dispersed

model systems. Many studies focused on the characterization of interfacial properties but

did not attempt to connect these with the stability of emulsions and/or foams. It is well-

known that these systems are considerably more complicated than the idealized experi-

mental setup in interfacial measurements. Therefore, interfacial properties can only be

indicator for behavior in dispersed systems.

One part of this dissertation was to characterize interfacial properties, like adsorption

and interfacial rheology of various saponins from different botanical origins and link these

properties to foam properties (manuscript I). The second part of the dissertation focused

on the interactions of Quillaja saponin (QS) and β-lactoglobulin (β-LG) in bulk (manu-

script II). Afterwards interactions of QS and β-LG were characterized at the air/water-

(manuscript II) and oil/water-interface (manuscript III) and these results were connected

to foam and emulsion properties, respectively. In the last part of the dissertation the prop-

erties of foamed emulsions were characterized, which contained oil, water and air

(manuscript IV). In these experiments, key factors were determined, which affected sta-

bility of foamed emulsions. Oil droplet size, pH and presence β-LG were identified as such

key factors.

In the following chapters the results of this thesis are discussed in a general way and the

light of new research findings. All subsections start with a short summary of the experi-

mental findings.

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6.1. The impact of structural features on interfacial properties of sapo-

nins from different botanical sources

In manuscript I, six different saponin-rich extracts from various botanical origin were ana-

lyzed with respect to adsorption as well as foaming, foam stability and foam structure

(summarized as foam properties). Saponin extracts were chosen based on a previous study

(Golemanov et al. 2013), which examined interfacial shear rheology of 13 saponin extracts.

In Figure 1 differences in molecular structure of the discussed saponins are visualized. In

the cited study, the authors grouped the saponin extracts based on their interfacial behavior.

For experiments in manuscript I, three extracts from group EV, which formed strong vis-

coelastic interfacial layers, were chosen: Quillaja saponaria Molina (QS), Camellia

oleifera Abel (TS) and Aesculus hippocastanum (ESC). To characterize the properties of

saponins, which showed no viscoelastic properties (group LV) two extracts from Tribulus

terrestris (TT) and Glycyrrhiza glabra (GA) were chosen as well as a previously not char-

acterized extract from Gypsophia (GYP). From the six saponin extracts five had a

triterpenoid aglyone and from these five QS and GYP mainly contain bidesmosidic sapo-

nins (two linked sugar residues) while TS, GA and ESC consist of monodesmosidc

saponins (one linked sugar residue). In contrast, TT is a mixture of mono- and bidesmosidic

saponins, which have a steroidal aglyone. In addition, conductivity and FTIR measure-

ments were performed to support results on foam stability due variation of pH and ionic

strength. Although the number of samples in this thesis was relatively small, it can be con-

cluded that usually, high dilational and shear viscoelasticity of saponin films at the

air/water-interface led to a considerably high foamability and foam stability. But it was also

shown that saponins with the highest dilational and shear moduli not necessarily yield the

highest foam stability. However, adsorption and interfacial configuration of saponins did

not correlate with foaming and foam stability. In addition, the classification of saponins as

ionic or non-ionic surfactants is not useful with respect to forecast sensitivity to changes in

pH and ionic strength.

It was shown that adsorption of all analyzed saponins is mixed-barrier controlled, which

is in agreement with a previous study (Wojciechowski et al. 2011). The calculated diffusion

coefficients were based on assumed hydrodynamic radii of the saponins. Calculated diffu-

sion coefficients were distinctly lower than experimental diffusion coefficients (see Table

I-3). This discrepancy led to the conclusion that adsorption is slowed down by an additional

General discussion

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barrier, which may be caused by steric hindrances and rearrangements of the saponin mol-

ecules upon adsorption. The amount of linked sugar residues may be an additional factor,

which influences adsorption. As illustrated by the parameter Π5s/Π1800s (see Table I-3) the

maximum surface pressure is reached faster by monodesmosidic saponins in comparison

to bidesmosidic saponins. It may be hypothesized that the additional sugar residue in

bidesmosidic saponins slows down adsorption.

As discussed in greater detail in chapter 2.3.1 and visualized in Figure 5 saponins may

obtain two different interfacial configurations: lay-on and end-on configuration indicated

by an area per molecule above and below 0.75 nm², respectively (Pagureva et al. 2016).

The area per molecule may be calculated by fitting the Frumkin-model to the concentration-

dependent interfacial tension values. In manuscript I, the determined interfacial area for

GYP indicated a lay-on configuration as expected for bidesmosidic saponins. For all other

saponins with the exception of TT the obtained interfacial area indicated an end-on config-

uration. Results from the adsorption isotherm for TT were ambiguous and did not clearly

indicate a certain type of interfacial configuration. As mentioned before, TT is a mixture of

mono- and bidesmosidic saponins, which may explain the ambiguous results. However,

interfacial configuration of saponins neither influenced adsorption nor foam properties.

When comparing the interfacial shear and dilational results of Golemanov et al. (2013)

and Pagureva et al. (2016) with the foam properties presented in Figure I-2 of manuscript

I it can be concluded that saponins with high dilational and shear viscoelasticity can form

stable foams. Thereby it was possible to produce a foam of all aqueous solutions containing

triterpenoid saponins. Bidesmosidic saponins (GYP and QS) yielded foams with highest

stability. In contrast, foam properties are especially low for steroid saponins (here: TT) and

saponins with only few sugar residues (here: GA), which also only showed a viscous re-

sponse in shear experiments of Golemanov et al. (2013).

It was shown by Pagureva et al. (2016) that monodesmosidic saponins (TS and ESC)

can undergo phase transition (indicated by high interaction values) and can form very high

elastic interfacial films, which are somewhat sensitive to dilational stress but rather insus-

ceptible to shear stress. The authors additionally demonstrated that Quillaja saponins

(bidesmosidic with long sugar chains and fatty acyl residue) can also form a strong visco-

elastic network, which is less shear viscoelastic but is less affected by dilational stress. The

reported phase transition may additionally contribute to the high foam stability of the ana-

lyzed saponin extracts TS and ESC.

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Relatively high instability of foams from GA can be attributed to the proportional low

amount of sugar residues in this saponin. The saponin is poorly soluble and only few inter-

molecular hydrogen bonds are formed between sugar residues, which consequentially lead

to a weak interfacial network as shown by creep-recovery experiments (Golemanov et al.

2013).

Although differences between triterpenoid and steroidal aglycone structure are relatively

small, these discrepancies tremendously impact interfacial rheology and foam properties.

The exact reason or structural feature of steroidal saponins, which leads to poor foam prop-

erties and viscous films, is yet to be determined. The low ability of TT to form an interfacial

viscoelastic network resulted in highly instable foams even at large concentrations of about

2 %. The very low ability to stabilize foams even at high concentrations, further supports

the hypothesis that interfacial packaging of steroidal saponins is disadvantageous and the

resulting interfacial film cannot withstand dilational and shear stress.

In colloidal science, low-molecular weight surfactants are usually classified as ‘ionic’

or ‘non-ionic’ based on their molecular structure. The former term is used when chargeable

groups are present in the molecular structure of the surfactant. The latter term refers to

surfactants with no chargeable groups and which interfacial properties are therefore insus-

ceptible to changes in ionic strength and changes in pH. In the past it was debated whether

saponins may be classified as ionic or non-ionic surfactants (Wojciechowski 2013, Feng et

al. 2015). In experiments of the present study the variation of pH and ionic strength showed

that classifying saponins as ionic or non-ionic is not useful. FTIR and conductivity experi-

ments revealed that QS, GYP, ESC and GA probably possessed chargeable groups or/and

ionic residues in the extract. But interfacial tension and foam stability of these extracts were

not always affected by changes in pH and ionic strength (see Figure I-3, Figure I-5 and

chapter I-4.4). On the other hand, interfacial tension and foam stability of TS, which was

classified as ‘non-ionic’, were tremendously influenced by changes in pH and ionic strength

(see Figure I-6).

Manuscript I was an important step to increase knowledge on the link between interfa-

cial rheology, adsorption and foam properties of saponins. However, due to diversity of

saponins the impact of structural features, like subcategory of aglycone structure (e.g.

oleanane, dammarane, ursolic to name a few), length of sugar residues and type of sugars

on interfacial properties is yet to be determined.

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6.2. Synergistic and antagonistic effects of QS/β-LG-mixtures on

stability of dispersed systems

As discussed in manuscript I and chapter 6.1, QS can thoroughly stabilize foams and is to-

date the best characterized saponin extract. Therefore, all following experiments (manu-

script II-IV) were performed with QS. The characterization of interfacial properties of an

aqueous solution of QS was the first step to understand the relationship between interfacial

properties, molecular structure and foam properties. In these relatively simple systems it is

possible to study correlations between interfacial properties and foam and/or emulsion

properties. Matrix effects occur when various constituents are mixed (proteins, lipids, car-

bohydrates, phenols and surfactants) and are a common phenomenon in food, animal

nutrition or pharmaceutical products. Interactions between surfactants and proteins are

well-known and were extensively researched in the past (Bos and van Vliet 2001; Pradines

et al. 2009; Lech et al. 2014). As there have been evidences supporting the hypothesis that

QS and β-LG form complexes, which influence interfacial properties (Kezwon and

Wojciechowski 2014), additional experiments are carried out in this thesis on the binary

system of QS and β-LG. In the next two subsections the interactions of QS with common

food protein β-lactoglobulin in foams (see chapter 6.2.1), emulsions (see chapter 6.2.1) and

foamed emulsions (see chapter 6.2.3) are discussed. All chapters start with a brief summary

of the results from each manuscript and afterwards interfacial behavior and corresponding

properties in dispersed systems are discussed.

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6.2.1. Synergistic effect of QS/β-LG-mixtures on foam properties

In manuscript II the interactions between QS and β-LG at the air/water-interface were char-

acterized with respect to interfacial shear and dilational rheology, adsorption, complex

formation and foam properties. The results of the experiments supported the hypothesis on

molecular interaction of QS and β-LG in the bulk as well at the air/water-interface. Due to

similar foam stabilizing mechanisms, a synergistic effect on foam stability was observed

when QS and β-LG were mixed.

Interactions between QS and β-LG in the bulk were observed in an experiment on fluo-

rescence quenching. Results indicated the formation of a ground-state complex (static

quenching) and the inverse slope of the Cogan-plot revealed that QS interacts with 1.7

binding sites of β-LG (see Figure II-1). The interactions between QS and β-LG might be

hydrogen bonds, electrostatic or hydrophobic interactions. In an additional experiment, it

was shown that QS may slowly displace β-LG from the air/water-interface (Figure II-2).

Whether displacement originated from the ‘orogenic displacement’ or the ‘competitive ad-

sorption and complexation’ mechanism is unclear. But as QS was classified as an ionic

surfactant (see Table I-5, Figure I-3 and chapter 6.1) it may be hypothesized that the ‘com-

petitive adsorption and complexation’ mechanism is more likely to occur (Kotsmar et al.

2009).

Experimental results on short-term adsorption (Figure II-3) and dynamic interfacial ten-

sion (Table II-1) of mixtures of QS and β-LG were similar to QS, which imply that the

interfacial film properties are dominated by QS. Interfacial interactions between QS and β-

LG were characterized using dilational and shear experiments. High values of the complex

viscoelastic moduli (E*>100 mN/m) were obtained when mixed interfacial films of QS and

β-LG were subjected to dilational stress (see Figure II-4). As reported before (Stanimirova

et al. 2011), QS film was highly viscoelastic with a high elastic proportion. All mixed in-

terfacial films exhibited a primary elastic response and viscous moduli were very low. The

high values for E* are a sign for intermolecular interactions between adsorbed molecules.

As all samples, QS, β-LG and their mixtures, exhibited high E* it can be concluded that

intermolecular interactions can be found in all interfacial films. These results were further

supported by interfacial shear experiments (see Figure II-5). High values for the complex

shear modulus (G*) up to 70 mN/m were determined for QS and β-LG-interfacial films.

Mixtures of QS and β-LG yielded even higher values for G*, which imply an increase in

intermolecular interaction. It was hypothesized that small concentrations of β-LG

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strengthen the QS network, in accordance to hypothesis 2, which was reported before for

another ionic surfactant and β-LG (Gunning et al. 2004). At higher β-LG concentration

(>0.05 %) this effect is neglected.

As presented in manuscript I, stables foams are formed by QS due to the viscoelastic

network. The stabilization of foam lamellae by a viscoelastic film is very unusual for such

a small surfactant (~2kDa) and is much more common for globular proteins. Usually low-

molecular weight surfactants (<0.5 kDa) stabilize foam films by the Gibbs-Marangoni

mechanism (Wilde et al. 2004) and are not able to form a viscoelastic network at the inter-

face. Viscoelastic moduli for low-molecular surfactants are usually low with E* around

10 mN/m as reported for SDS (Fainerman et al. 2010). For the Gibbs-Marangoni mecha-

nism lateral mobility of the adsorbed surfactant molecules is necessary. Gravitational forces

cause steady drainage of foam lamellae, thus decreasing their thickness. Concentration and

interfacial tension gradients in the foam lamella, cause molecules from surfactant-enriched

regions move along the interface to level out differences in concentration and interfacial

tension. Hereby bulk solution is dragged along simultaneously and thereby restoring thick-

ness of the foam lamellae. Quillaja saponins cannot stabilize foam lamellae by the Gibbs-

Marangoni mechanism because intermolecular bonds fix saponin molecules inside the vis-

coelastic network.

Confirming hypothesis 2, in foam experiments it was shown that mixing QS and β-LG

had a beneficial effect on foam density and foam stability. Even a small β-LG concentration

tremendously increased foam stability (see Figure II-6). Foam stability distinctively in-

creased in mixed QS/β-LG-system because both substances form a strong joint viscoelastic

network, which stabilizes foam lamellae (see Figure 9D). This is different from other pro-

tein-surfactant systems: usually foam stability in mixed systems is reduced because

surfactants stabilize foam lamellae by the Gibbs-Marangoni mechanism, which requires

high lateral mobility (Maldonado-Valderrama and Patino 2010). On the one hand, in pro-

tein-surfactant systems the lateral movement of the surfactant molecules is blocked by

adsorbed protein molecules (see Figure 9A-C). On the other hand, the viscoelastic network

of proteins is weakened by surfactants molecules.

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Figure 9A-C) Vulnerability of thin films stabilized with a mixture of surfactant and protein

(Adapted from Wilde et al. 2004 with permission from Elsevier) and D) joint interfacial

network of QS and β-LG with intermolecular interactions (red dotted lines)

In manuscript II, results were presented supporting the hypothesis that complexes and in-

teractions between QS and β-LG may occur both in the bulk and at the air/water-interface.

These interactions did not only increase viscoelastic moduli in interfacial experiments but

also foam stability was considerably increased in mixtures of QS and β-LG. Synergistic

properties in surfactant/protein-systems are unusual and may open new opportunities in

applications.

Protein interactions weakened

Surfactant fluidity limited

Protein interactions break down

Surfactant migration hindered

A) Mixed film

C) Rupture

B) Film deformation

Protein

Surfactant

β-LG

General discussion

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6.2.2. Antagonistic effect of QS/β-LG-mixtures on emulsion stability

In manuscript II interactions of QS and β-LG in aqueous bulk as well as synergistic prop-

erties at the air/water-interface and in foams were determined. In contrast, experimental

work in manuscript III was performed to investigate QS/β-LG-interaction at a hydrophobic

phase containing triglycerides (here: MCT-oil). It was shown, using similar experimental

methods like in manuscript II that interactions between QS and β-LG lead to a viscous film

that was prone to non-linear behavior upon imposed stress. In emulsions, mixtures of QS

and β-LG led to extensive aggregation, which reduced kinetic stability.

The interfacial film containing QS was distinctly less viscoelastic and had a higher viscous

proportion compared to the air/water-interface as visualized in Figure III-2,3,4 and 5. The

reduction of viscoelastic properties of QS-films was described before by several authors

(Wojciechowski 2013; Golemanov et al. 2014). In contrast, interfacial layers of β-LG were

less affected by changes of the hydrophobic phase and remained primary elastic (indicated

by low Φ in Figure III-3).

However, it remains unclear whether configuration of QS molecules adjusts when the

hydrophobic phase is changed from air to oil. Several studies reported data, which showed

that the area per molecule of a QS extract increased from 0.43 nm² at the air/water-interface

(Böttcher and Drusch 2016) to ~1 nm² at the MCT-oil (Yang et al. 2013; Tippel et al.

2016b) and corn oil/water-interface water (Bai et al. 2016). But Wojciechowski (2013)

presented results on the area per molecule for a Quillaja saponin extract, which did not

differ at the air/water and tetradecane/water-interface. The increase in interfacial area per

molecule from the air/water- to the triglyceride/water-interface may be associated with a

change in configuration from end-on to lay-on configuration (see chapter 2.3.1) or may

simply be attributed to the penetration of oil between adsorbed QS molecules (see chapter

2.3.3).

As discussed in further detail in chapter 2.3.3 linear alkanes and triglycerides may pen-

etrate between adsorbed saponin molecules. Especially bulky triglycerides enter between

adsorbed saponin molecules to hydrate the polar head groups and decreases intermolecular

interactions between neighboring saponin molecules (see Figure 8). It may be therefore

possible that the linear alkane tetradecane did lead to an increase in area per molecule as

observed at the corn oil and MCT oil/water-interface in comparison to the air/water-inter-

face, because tetradecane is less bulky.

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Another reason may be a difference in adsorbed interfacial active residues of the saponin

extract. It was characterized by Tippel et al. (2017) that phenols in the QS extract differ in

hydrophilicity. Thus, with differing hydrophobicity of the non-aqueous phase other non-

saponin constituents may adsorb at the interface and lead to differences in molecular ar-

rangement at the interface.

Similar to manuscript II, experimental results of manuscript III showed that properties

of mixed interfacial films containing QS and β-LG at the oil/water-interface were domi-

nated by QS. Mixed interfacial layers of QS/β-LG showed a non-linear response when

subjected to dilational stress as indicated by Lissajous-plots (see Figure III-4). From Lissa-

jous-plots it may be derived that the interfacial network between QS and β-LG is relatively

stiff and is not able to rapidly adjust to increase of droplet volume (see Figure III-5). Stiff-

ness of the interfacial network of mixed QS/β-LG films decreased with increasing β-LG-

concentration. Shear experiments supported the hypothesis on lateral interactions between

QS and β-LG at the interface due to high shear moduli.

The divergent interfacial behavior of mixtures of QS and β-LG at the oil/water-interface

in comparison to the air/water-interface may be attributed to differing properties of the in-

dividual substances. It is well-known that β-LG exhibits different adsorption kinetics at the

oil/water-interface in comparison to the air/water-interface (Zhai et al. 2013; Zare et al.

2016). It is believed that unfolding is more pronounced at the oil/water-interface and there-

fore interfacial properties vary distinctly.

In further experiments in which emulsion properties were characterized it was shown

that mixed QS and β-LG-emulsions were stabilized by electrostatic repulsion (see Figure

III-8). The high negative charge of oil droplets covered by QS was reported before and

attributed to the carboxylic group of QS and anionic residues like (+)-piscidic acid, syringic

acid and p-coumaric acid, which can found in the crude extract (Maier et al. 2015a; Tippel

et al. 2017). It was shown that purification and thereby removing anionic non-saponin res-

idues, can increase absolute values of ζ-potential from -70 mV to -50 mV (pH 7). β-LG is

negatively charged at pH 7 because this pH is above the isoelectric point, which is around

5.1 (Schwenke 1998).

In mixed QS/β-LG-emulsions with high β-LG-concentrations considerable aggregation

was observed, which led to intense creaming after storage time of 7 days. Aggregation was

attributed to partial displacement and/or structural changes of β-LG induced by QS. It is

General discussion

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possible that QS induces changes in the tertiary or quaternary structure of β-LG when com-

plexes are formed. Prompted structural changes of β-LG by a phenol were described before

for green tea polyphenol/β-LG-systems (Staszewski et al. 2014). It may also be possible

that, as reported in manuscript II, QS partially desorbs β-LG and that the detached β-LG

parts cause the aggregation.

In summary, manuscript III revealed that properties of mixed interfacial films of QS and

β-LG at the oil/water-interface are considerably different from the air/water-interface. Re-

sults indicate that interfacial interactions lead to a stiff network which is prone to non-linear

behavior upon imposed stress. Furthermore, QS induced changes of β-LG, which prompted

aggregation of oil droplets, thus reducing kinetic stability of emulsions.

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6.2.3. Maximizing stability of foamed emulsions

Aim of manuscript IV was to evaluate important factors that affect stability of the foamed

emulsions by using knowledge on destabilizing mechanism of foams and emulsions as well

as finding from previous studies of this thesis (manuscript II+III). Foamed emulsions are a

more complex dispersed system in comparison to foam and emulsion, which were de-

scribed in manuscript II and III, respectively. It was hypothesized and shown that emulsions

with a moderate oil droplet size yielded foamed emulsions with the highest stability. Fur-

thermore, with increasing pH, electrostatic charge of oil droplets and in thin liquid films

increased, which contributed to increased stability of the foamed emulsions. Another tool

for increasing the stability of foamed QS emulsions is the use of β-LG to stabilize the

oil/water-interface while QS stabilizes the air/water-interface.

Dispersed oil droplets in foams may be referred to as antifoams and either lead to a fast

or slow foam destruction depending on the distribution of the oil droplets in the foam. The

thermodynamic conditions, which determine the location of the oil droplets either in the

Plateau borders (slow antifoam) or the thin liquid film (fast antifoam) may be determined

using the film trapping technique (FTT). With the FTT the critical pressure of rupturing of

an A/W/O-pseudoemulsion film is measured, which is also called ‘entry-barrier’. Fast an-

tifoams have a low entry-barrier and slow antifoams a high entry-barrier (Denkov 2004;

Miller 2008). It was concluded that dispersed MCT-oil may be classified as a slow antifoam

(see Figure 10) because foam decay was relatively slow (see Figure IV-5). Therefore, it

was hypothesized that oil droplets accumulate in Plateau borders because slow antifoams

cannot enter the thin liquid film due to thermodynamic conditions (Karakashev and Groz-

danova 2012). Foam destruction in this case originates from liquid drainage, which reduced

size of the Plateau borders and localized oil droplets subsequently lead to film rupture by

spreading and bridging the Plateau borders. In contrast, fast antifoams may enter the thin

liquid film between two adjacent bubbles and lead to bridging of foam lamella within sec-

onds.

The influence of the oil droplet size on the stability of the foamed emulsion was shown

by preparing emulsions with a constant QS-concentration and varying oil droplet size with

a d50 ranging from 0.2 to 2 µm. At the optimal oil droplet size (0.6-1 µm), stability of the

foamed emulsion was maximized (see Figure IV-4 and Figure IV-5). When oil droplets

were smaller than the optimal size the amount of free QS was not sufficient to cover the

oil/water- and air/water-interface at the same time and as a result foamed emulsions were

General discussion

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Sandra Böttcher Technische Universität Berlin

highly instable. Due to the insufficient coverage of the freshly formed bubble surface by

QS rapid bubble coalescence occurred. The bubble coalescence during foaming led to a

very low foaming speed. In contrast, large oil droplets have a proportionally smaller inter-

facial area and QS can stabilize both interfaces, which led to high foamability of emulsions.

Free QS, which is not adsorbed at the oil/water-interface, adsorbs at the air/water-interface.

The adsorption lowers the interfacial tension and a viscoelastic film is formed, which slow

down thin liquid film rupture and coalescence. But the increased size of the oil droplets

simultaneously accelerated bridging thin liquid films, which decreased foam stability.

Figure 10 Dispersed droplets of MCT-oil presumably accumulate in Plateau borders as it is char-

acteristic for slow antifoam

In experiments with varying pH it was shown that stability of the foamed emulsions was

pH dependent and was maximized at pH 7 (Figure IV-7). The pH dependency of the sta-

bility of the foamed emulsions may be attributed to anionic residues present in the QS

extract as well as the carboxylic group in the molecular structure of QS. Various anionic

residues were reported before (Maier et al. 2015; Tippel et al. 2017), which affect the elec-

trostatic conditions in the thin liquid film and on the surface of oil droplets. At very low pH

(here: pH 2) electrostatic charge of the dispersed oil droplets was low (indicated by ζ-po-

tential close to zero, see Figure IV-8). Therefore, electrostatic repulsion was too low to

prevent coalescence of oil droplets, which caused distinct size enhancement of the oil drop-

lets. Thus, foamed emulsions at pH 2 were highly instable due to the size of the aggregates,

which accelerated bridging of foam lamellae. With increasing pH the electrostatic repulsion

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General discussion

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between oil droplets increased (as indicated by ζ-potential), which prevented aggregation

of oil droplets, thus increasing stability of the foamed emulsions. Stability of the foamed

emulsions increased above pH 5 although ζ-potential did not change. It was therefore hy-

pothesized that anionic residues may adsorb at the air/water-interface and that further

increase of pH led to further deprotonation of the anionic groups. Thus electrostatic repul-

sion in the thin liquid films increased. At increasing pH electrostatic repulsion in the thin

liquid film opposes further thinning, which increases foam stability.

In thin liquid films (<100 nm) attractive and repulsive forces determine the stability of

the film. These forces may be repulsive electrostatic, attractive van der Waals and repulsive

steric forces. The disjoining pressure Π thereby sums up the condition in the thin liquid

film. When the disjoining pressure is smaller than the capillary pressure (which originates

from pressure differences between the dispersed and continuous phase), the thin liquid film

is thinning. With decreasing thickness of the thin liquid film the disjoining pressure changes

and meta-stable states like common black films and Newton black films may be obtained.

(Stubenrauch and von Klitzing 2003; Fauser and von Klitzing 2014; Schramm 2005)

In food products, proteins are commonly present and it was shown before in manuscript

II and III that QS and β-LG interact in the bulk and at the interface. In manuscript IV the

impact of β-LG on the stability of foamed QS-emulsions was characterized. Based on pre-

viously reported results on interfacial rheology it was hypothesized that QS is most suitable

to stabilize the air/water-interface and β-LG is best in stabilizing the oil/water-interface.

Results of interfacial tension, ζ-potential and foaming speed showed that this distribution

of QS and β-LG can be obtained by sequentially adding QS to a β-LG emulsion (see Figure

IV-10). As hypothesized it was shown that the stability of a foamed QS/β-LG emulsion

distinctly increased by sequential addition of QS to a previously homogenized β-LG emul-

sion (Mixpost). In contrast, when QS and β-LG were simultaneously homogenized stability

of a QS/β-LG emulsion was considerably lower. Mixed films of QS and β-LG are on the

one hand beneficial at the air/water-interface and mixed films showed higher viscoelastic-

ity, which resulted in higher foam stability (as shown in manuscript II). On the other hand

mixtures of QS and β-LG at the oil/water-interface led to interfacial layers, which re-

sponded to interfacial stress (dilational experiments) with non-linear behavior and had a

high viscous proportion (manuscript III). Apart from that QS/β-LG-emulsions were prone

to aggregation, which decreased emulsion stability. In foamed emulsions (Mixpre) mixed

interfacial layers of QS and β-LG at the air/water- and oil/water-interface did not increase

General discussion

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Sandra Böttcher Technische Universität Berlin

stability of the foamed emulsions in comparison to a foamed QS-emulsion. It was expected

that stability of foamed emulsions would increase when β-LG is added, because the amount

of surface active molecules was increased.

In manuscript IV findings of the previous manuscripts were applied to determine major

factors, which impact stability of a food product-like system (foamed emulsions). Oil drop-

let size was a crucial factor to control stability of the foamed emulsions. It was shown that

a moderate oil droplet size should be applied to minimize bridging foam lamella by oil

droplets and to maximize amount of free surfactant. Stability of the foamed emulsion may

also be increased by adjusting a high pH, which increased electrostatic repulsion in thin

liquid films and between oil droplets. Furthermore the oil/water-interface should be stabi-

lized by another surface active constituent (here: β-LG) in order for QS to solely stabilize

the air/water-interface.

Concluding remarks and outlook

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Concluding remarks and outlook

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7. Concluding remarks and outlook

The comprehensive experimental methodology of the present thesis led to a significant

contribution in understanding of the structure-function relationship in saponin-based dis-

persed systems. This thesis aimed to connect behavior of saponins at aqueous interfaces

with foam and emulsion properties. It was further aimed to explain underlying stabilizing

or destabilizing phenomena of dispersed systems containing Quillaja saponin (QS) and β-

lactoglobulin (β-LG).

The present work showed that there are several structural features of saponins, which

are associated with good foam properties (high foaming speed, liquid content and foam

stability). Saponins should have a triterpenoid aglycone structure of oleanane type. The

amount of sugar residues seems to play a minor role on interfacial properties, but as dis-

cussed, sugar chains should have a certain length to provide sufficient hydrophilicity. The

good foam properties of triterpenoid saponins were attributed to the formation of a highly

viscoelastic network at the air/water-interface through intermolecular hydrogen bonds be-

tween neighboring sugar residues. It was therefore concluded that saponin foams are not

stabilized by the Gibbs-Marangoni mechanisms, which usually applies for low-molecular

weight surfactants.

It was shown that QS has unique properties at aqueous interfaces and in dispersed sys-

tems. QS is suitable to stabilize foams and emulsions by the formation of a viscoelastic

network and electrostatic repulsion, respectively. High foam stability was supported by data

on interfacial rheology from previous publications, which revealed unusually high viscoe-

lastic moduli.

In mixtures of QS and β-LG, complex formation in the bulk and interactions at aqueous

interfaces (here: air/water and MCT-oil/water) were shown by fluorescence experiments

and pendant drop experiments as well as shear rheology, respectively. It was further dis-

cussed that QS desorbs β-LG from the air/water-interface and it was assumed that this was

due to the competitive adsorption and complexation mechanism. Foam stability distinctly

increased in mixed QS/β-LG-systems due to the joint viscoelastic network formed by both

constituents. The usual antagonistic effect (competitive stabilization by Gibbs Marangoni

mechanism vs. the viscoelastic network) of mixing of a low-molecular weight surfactant

and protein on foam properties was not observed.

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Results furthermore showed that interfacial properties of mixed QS/β-LG-systems were

distinctly affected by the type of the hydrophobic phase. In contrast to the air/water-inter-

face interfacial layers at the oil/water-interface were distinctly less viscoelastic and had a

high viscous proportion. These differences were attributed to changes in interfacial arrange-

ment of QS and β-LG due to changes of hydrophobicity of the non-aqueous phase. The

aggregation of oil droplets in emulsions containing QS and β-LG presumably originated

from structural changes of β-LG (tertiary or quaternary structure) induced by QS and/or

partial displacement of β-LG from the oil/water-interface.

This thesis showed that stability of complex systems like foamed emulsion may be con-

trolled by a few key factors. The oil droplet size and the pH of an emulsion are crucial

factors that affect stability of the foamed emulsion. Oil droplets should be in a medium size

to postpone bridging of oil droplets and to provide sufficient amount of free surfactant that

can stabilize the air/water-interface. At high pH the stability of the foamed emulsions is

highest due to electrostatic repulsion of oil droplets, which slows down shrinkage of Plateau

borders. In addition, the higher electrostatic repulsion in foam lamellae also reduces drain-

age of the aqueous phase from the thin liquid film and therefore postpones rupture of foam

lamellae. Based on the speed of defoaming it was concluded that the dispersed MCT-oil

may be classified as a slow antifoam and may therefore be located in the Plateau borders

of the foam.

From an academic point of view it would be interesting in future studies to deepen un-

derstanding on the relationship between molecular features of saponins and their interfacial

properties by performing experiments with a larger samples size including plants without

commercially available extracts. Preparative HPLC methods can help to obtain purified

saponin extracts. These studies should especially include saponins with aglycone structures

different from oleanane type like ursolic and dammarane type. In addition, the role of the

length of sugar residues and other structural features like fatty acyl group should be exam-

ined. It would be expected (as stated in this thesis) that with increasing length of the sugar

residues (regardless of mono- or bidesmosidic) the hydrophilicity increases, which leads to

higher stability of foams. Future project should involve close cooperation of analytical

chemists and research groups focusing on interfacial properties. When reliable information

on chemical composition of saponin extracts are available and are directly connected to

Concluding remarks and outlook

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Sandra Böttcher Technische Universität Berlin

interfacial properties, even more distinct and meaningful correlations on structure-function-

relationship can be obtained.

Further research should also examine the supramolecular structure of monodesmosidic

saponins and observed phase transition of some monodesmosidic saponins. More funda-

mental studies may address the inability of steroid saponins to form viscoelastic layers in

more detail to identify the responsible molecular feature. One major question is whether

and how the level of viscoelasticity is connected to foam properties. Besides that, compre-

hensive data are still necessary to also connect interfacial properties with emulsifying and

emulsion stabilizing properties as viscoelasticity of the interfacial layers may distinctly af-

fect emulsion properties.

The knowledge on mixtures of QS and β-LG distinctly increased in the last years and

the analysis of other proteins, like random coil proteins would be an interesting opportunity

due to the absence of tertiary and quarternary structure. Another scope of future research

project may be the mechanism by which QS desorbs β-LG and other proteins. Therefore,

sequential adsorption of QS to a loaded β-LG interface may be studied using Brewster angle

microscopy or atomic force microscopy (AFM). These methods were successfully applied

in previous studies for other surfactant/protein-systems. To better understand stabilizing

mechanisms in QS/β-LG foams model experiments of thin liquid films should be performed

by thin film pressure balance experiments. In this context, it would be interesting to deter-

mine whether QS and its mixtures with β-LG are able to form common black films or

Newton black films. These terms refer to thermodynamically meta-stable states of thin liq-

uid films and are associated with high stability. The interfacial arrangement of QS and β-

LG (or other proteins) at aqueous interfaces may be analyzed by further studies by ellip-

sometry, which gives insights in thickness of the interfacial layer. To determine the

underlying mechanism that is responsible for the aggregation of oil droplets in mixed QS/β-

LG emulsions, Fourier transform infrared spectroscopy may be used to determine structural

change of β-LG.

From an industrial point of view, the interactions between QS and other (food) constit-

uents, like inorganic salts and hydrocolloids need to be determined to control properties of

dispersed systems. Salts are well-known to affect electrostatic charge of functional groups

and a screening of these groups may reduce stability of dispersed systems because electro-

static repulsion between oil droplets and in thin liquid films may be reduced. To enhance

knowledge on the underlying mechanisms that are responsible for the stability of foamed

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emulsions fundamental studies may be connected to stability tests. One option for a funda-

mental approach is the film trapping technique, which may be used to determine the entry

barrier of dispersed MCT-oil droplets and to derive the location of the oil droplets in the

foam. For visual conformation of the location of the oil droplets light microscopy may also

be used.

With respect to sustainability and ecological responsibility, research projects should fo-

cus on saponins from sustainable resources like legumes. Saponins are undesired

constituents in animal nutrition due to adverse gastro-intestinal effects. By extracting sap-

onins and using them in food products the added value of legumes increases and financial

incentive may increase popularity of legumes for agricultural businesses. Saponins from

legumes are usually monodesmosidic saponins and in the case of pea saponins an additional

heat-sensitive group is linked to the aglycone structure. It is yet unclear how the functional

group affects interfacial properties and if these saponins may be used in dispersed systems.

It can be summarized, that saponins, despite of some drawback, are a promising class of

natural surfactants to be used in dispersed systems. The haemolytic activity of saponins is

an issue when using saponins as adjuvants in vaccines, but low dosage of saponins in food

should not cause adverse effects on human health. Molecular structure of saponins is nei-

ther similar to low-molecular weight surfactants nor globular proteins and the same holds

true for interfacial properties and stabilization mechanisms in foams. But a generalization

of the properties of saponins is difficult due to the immense structural diversity. A vision

for the future may be the customization of interfacial properties by mixing various saponin

derivates. In addition, the use of sustainable resources is an excellent option to increase

added value of low-price products like legumes.

References

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Annex

A-I Materials and devices

Table A-1 Origin, purity and general chemical properties of the used saponin extracts

Material Supplier Purity [%]

Extract from Quillaja saponaria Molina

(QS)

Ingredion Germany GmbH 69.2

Extract from Gypsophila (GYP) Dr. H. Schmittmann GmbH 45.5

Extract from Camellia oleifera Abel

(TS)

Changsha Nulant Chem. Co., Ltd. 95.3

Extract from Aesculus hippocastanum Sigma Aldrich Chemie GmbH 99.1

Extract from Glycyrrhiza glabra Sigma Aldrich Chemie GmbH 95.1

Extract from Tribulus terrestris Xi'An Union Pharmpro Co., Ltd 90.4

Sodium dodecyl sulfate (SDS) Sigma Aldrich Chemie GmbH >99

Vanillin Carl Roth GmbH & Co. KG 100

Ethanol Carl Roth GmbH & Co. KG 99.5

Sulphuric acid Carl Roth GmbH & Co. KG 96

Whey protein isolate Fonterra DSE -

Native β-Lactoglobulin Isolated from whey protein isolate 89

KH2PO Carl Roth GmbH & Co. KG >99

K2HPO4 Carl Roth GmbH & Co. KG >99

Methanol VWR International GmbH >99

Ammonia Merck & Co. 32

Medium-chain triglyceride oil

(MCT-oil) WITARIX® MCT 60/40 CREMER OLEO GmbH & Co. KG

C8: 57.5%

C10: 41.8%

C12:0.6%

Florisil Carl Roth GmbH & Co. KG

MgO: 15.5 %

SiO2: 84 %

Na2So4:0.5 %

Oil red O / /

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Table A-2 Devices

Device

Manufacturer

Tradename

Foam device KRÜSS GmbH DFA 100

Glass frit DURAN Group GmbH FL4502 Filter Plate G4

Glass column / CY4502 Glass Column

Tensiometer with Wilhelmy

plate

KRÜSS GmbH K11

Conductometer

Mettler-Toledo GmbH

Seven Easy

Electrode Conductometer Mettler-Toledo GmbH InLab 731

Membrane for Dialysis SPECTRUM® LABORATORIES, INC

Spectra/Por 1 Dialysis Mem-

brane

Drop shape analysis DataPhysics Instruments GmbH OCA-20

Two-fluid channel needle Self-made -

Oscillation unit DataPhysics Instruments GmbH ODG-20

Dosing unit DataPhysics Instruments GmbH ES 6

1 mL syringe B.Braun Melsungen AG Injekt F

C-18 column

Thermo Fisher Scientific Germany BV

& Co KG

HyperSep C18 10g/75mL

FTIR Bruker Corporation Bruker Tensor 27

High pressure homogenizer Niro Soavi Deutschland Panda 2K

Particle Sizer Retsch GmbH & Co. KG LA-950 Horiba

Photometer

Thermo Fisher Scientific Ger-

many BV & Co KG

Helios Omega UV-VIS

Digital single-lens reflex

camera

Nikon GmbH 3100

Camera lens Nikon GmbH

AF-S Nikkor 18-55 mm

1:3.5-5.6G

Fluorescence

Spectrophotometer

Agilent Technologies Deutschland

GmbH &Co. KG

Cary Eclipse

Rheometer Anton Paar Germany GmbH Physica MCR301

Interfacial rheology system

cell

Anton Paar Germany GmbH /

Bicone tool Anton Paar Germany GmbH /

Zetasizer Malvern Instruments GmbH Nano Zetasizer ZS

Zeta cells Malvern Instruments GmbH DTS 1060 C

Ultra-Turrax IKA -Werke GmbH & CO. KG T25 basic

Microscope

Vacuum block Carl Roth GmbH & Co. KG 16 slots

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A-II Characterizing foaming, foam stability and foam structure

Foam experiments were conducted using the foaming device DFA 100 (KRÜSS GmbH,

Hamburg, Germany). For all experiments the following parameters were chosen: 50 mL of

a surfactant solution were filled in a glass column (Ø40 mm). Foam was generated by purg-

ing pressurized air (0.15 L/min) through a porous glass frit (pore size 40-100 µm). As soon

as the sum of liquid and foam reached 180 mm, foaming was stopped. Foam profiles were

recorded by transmissibility measurement every 0.5 s for 1 hour. During that time foam and

liquid height as well as the average brightness profile were measured and recorded. Figure

A-1 shows typical foam profiles of (A) a foam made from 0.01 % and (B) a foam made of

0.003 % saponin solution.

Figure A-1 Foam profile of a foam made from a (A) 0.003 % and (B) 0.01 % QS solution

In addition, photographs of the foam were taken every 10 min with a Nikon D3100 to have

visual information on foam structure. All experiments were conducted under light exclu-

sion to minimize external influences on the brightness profiles. The device was thoroughly

rinsed with distilled water between experiments. All foaming experiments were conducted

at least twice.

The foams were characterized using multiple parameters, see Figure A-2. All derived pa-

rameters were clustered in foaming, foam stability and foam structure.

time [s]

foam/liquid heigth [mm]

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Figure A-2 Derived parameters from foam profile 1 - Time to maximum foaming level – tfmax [s],

2 - Maximum foaming level – fmax[mm], 3 - Slope of foaming – kf [mm/s],

4 – Relative remaining foam height– fn%[%], 5 - Foam half-life time – tf1/2[s],

6 - Stability of maximum foam height – tfmax-5% [s], 7 – relative drainage – dn%[%],

8 - Foam density – fden,n[%], 9 - Foam density at f-max – fden,fmax% [%],

10 - Analysis of image brightness distribution – median BDm,n and width BDw,n

In Table A-3 all parameters, their abbreviation and measuring unit are displayed. If the

parameters were calculated from other parameters the calculation is also specified. The

subscript n represents variable time points. For example f1800s is the relative remaining foam

heigth after 1800 s. Foaming speed kf was calculated from the deviation of foam height in

relation to time from the start of the foaming until the maximum foam height (fmax). The

parameters maximum foaming level fmax and time to maximum foaming level tfmx were se-

lected from the data. The relative foam remaining foam heigth fn% characterizes the

percentaged amount of foam still present in relation to fmax. A high foam decay is indicated

by small fn% values. The foam half-life time tf1/2 is defined as the time when 50 % of the

foam collapsed. The stability of the maximum foam height tfmax-5% is defined as the time

from reaching fmax until 95 % that height is still present. The relative drainage dn% is de-

fined as the percentage of liquid draining from the foam in relation to the liquid height at

the fmax. The parameters relative foam density fden,n describe the percentage of incorpo-

rated liquid in the foam.

Δ % 5

Δ%, 4

phase I

phase II

Time [s]

liquid

foam

8, 9, 10

Foam/liquid height [mm]

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Table A-3 Parameters to characterize foaming, foam stability and foam structure; n - time point

during the analysis; / - none

Parameter Abbrevation Unit Calculation/Definition

foaming

Time to maximum foaming

level

tfmax s

𝑡𝑡(𝑓𝑓𝑚𝑚𝑐𝑐𝑚𝑚)

Maximum foaming level fmax mm

𝑓𝑓𝑛𝑛=𝑚𝑚𝑎𝑎𝑚𝑚

Foaming speed kf mm/s

𝛥𝛥𝑓𝑓

Δt;𝑓𝑓≤𝑓𝑓𝑚𝑚𝑐𝑐𝑚𝑚;𝑡𝑡≤𝑡𝑡(𝑓𝑓𝑚𝑚𝑐𝑐𝑚𝑚)

foam stability

Relative remaining foam

height fn% %

𝑓𝑓𝑛𝑛

𝑓𝑓𝑒𝑒𝑚𝑚𝑒𝑒 ∙100

;

𝑡𝑡>𝑡𝑡𝑓𝑓𝑚𝑚𝑐𝑐𝑚𝑚

Foam half-life time tf1/2 s

𝑡𝑡(0.5𝑓𝑓𝑚𝑚𝑐𝑐𝑚𝑚)−𝑡𝑡(𝑓𝑓𝑚𝑚𝑐𝑐𝑚𝑚)

;

𝑡𝑡>𝑡𝑡𝑓𝑓𝑚𝑚𝑐𝑐𝑚𝑚

Stability of maximum foam

height tfmax-5% s

𝑡𝑡(0.95𝑓𝑓𝑚𝑚𝑐𝑐𝑚𝑚)−𝑡𝑡(𝑓𝑓𝑚𝑚𝑐𝑐𝑚𝑚)

;

𝑡𝑡>𝑡𝑡𝑓𝑓𝑚𝑚𝑐𝑐𝑚𝑚

Relative drainage dn% %

𝑎𝑎𝑏𝑏𝑎𝑎�𝑑𝑑𝑛𝑛

𝑑𝑑(𝑓𝑓

max

)∙100�

;

𝑡𝑡>𝑡𝑡𝑓𝑓−𝑚𝑚𝑐𝑐𝑚𝑚

foam structure

Relative foam density fden,n %

𝑎𝑎𝑏𝑏𝑎𝑎�(𝑑𝑑0−𝑑𝑑𝑛𝑛)

𝑓𝑓n ∙100�

Median of brightness distri-

bution

BDm,n / See Figure A-3

Width of brightness distri-

bution

BDw,n / See Figure A-3

Determination of foam structure with analysis of brightness profiles and foam pictures

Since there was no device available to determine the bubble size distribution and variations

in foam density were very high, a new semi-quantitative method was established. For this

purpose the brightness profile generated by the foaming device was analyzed, see Figure A-

The brightness profile is generated from the transmissibility data of the column and the

foam device can derive liquid and foam height from the foam profile. Zones filled with air

or liquid have a high transmissibility and appear white in the brightness profile since light

is not scattered. When light is sent through foam, the light is scattered at the bubbles, which

leads to darker areas in the foam profile. That means, the smaller the bubble the more light

is scattered and the less light transmits through the foam and the darker is the area on the

foam profile. Each pixel on the Y-axis displays the average transmissibility at a certain

height of measurement and each pixel on the X-axis represents the average transmissibility

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Sandra Böttcher Technische Universität Berlin

at a specific time point. At different time points the brightness distribution within the foam

in the column was measured in an area of 1 px width, which corresponds to a specific time

point, using Adobe Photoshop CS6 Extended. The brightness distribution ranges from 0-

255. 0 represents black and 255 represents white. In this study, data points between 1 and

253 were used to eliminate white background color. In Figure A-3 the procedure is dis-

played. From the brightness distribution the median (BDm,n) as well as the width (BDw,n)

between the d10 (10 % of the brightness values are beneath this value) and d90 were calcu-

lated for individual time points n. The higher the BDm is, the more light is transmitted

through the foam indicating that the foam is less dense. An increase of BDw indicates that

the foam structure is less homogeneous.

Figure A-3 Example for histogram of brightness distribution at a specific time point (rectangle of

1 px width) of a foam made from 0.01 % QS solution after 1800 s

A-III Validation of foam analysis

The used method for characterization of foaming, foam stability and foam structure had to

be validated in order to evaluate the quality of the results. Therefore, the coefficients of

variation of the different parameters of two different foam types were analyzed: foams with

higher density and therefore higher content of liquid (Higher density foams) and foams

with lower density with a smaller amount of incorporated liquid (Lower density foams).

This discrimination was necessary because lower density foams had high and irregular

foam decay. In addition, difference of the liquid height at the time points ‘maximum foam

1 253

Amount of pixels [-]

Brightness value [-]

d10 d90

Median d

(BD

)

Width (BDw)

1 px

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Sandra Böttcher Technische Universität Berlin

height’ and ‘end of measurement’ was less than 2 mm. In higher density foams the differ-

ence was still low with about 10 mm but normal variation of the device had considerably

less impact on the results compared to low density foams.

In Table A-4 the coefficients of variation (CV) of all analyzed parameters are listed. Con-

centrations of 0.003 % (5 replicates) and 0.01 % (4 replicates) of QS were chosen as a

representative of low and high density foams, respectively. For parameters with the sub-

script n the CVs if of all measurement points (0, 10, 20 … 60 min) were averaged.

Table A-4 Variation of parameters to characterize foaming, foam stability and foam structure de-

pending on QS concentration; n - time point during the analysis

Parameter CV0.003% [%] CV0.01% [%]

foaming

tfmax 0.5 0.4

fmax 5.6 0.1

kf 2.4 1.0

foam

stability

fn% 5.2 0.6

tf1/2 11.1 n.a.

tfmax-5% 31.6 n.a.

dn% 42.6 18.3

foam

structure

fden,n 45.6 21.7

BDm,n 5.4 2.1

BDw,n 12.1 3.2

The parameters time to maximum foaming level tfmax and foaming speed kf are quite similar

and had only small CVs below 5 %. With both parameters it is possible to compare different

foaming behaviors, but it is not necessary to determine both parameters for each foam in-

dividually. In our further analysis only foaming speeds are compared. High values for the

maximum foaming level fmax indicate a higher foam density since the higher fmax is, the

more foam was produced and the more fluid was incorporated in the foam. However, more

sophisticated parameters are used in this work to quantify foam density and the amount of

liquid in the foam. For this reason we only compare foaming speeds of different foams to

characterize the foaming.

To characterize foam stability it is possible to compare several parameters. As a standard

parameter, the relative remaining foam height fn% at different time point n can be charac-

terized. It is also possible to compare the foam half-life time tf1/2 but for this parameter

foams have to collapse at least to 50 %, which is not applicable for most saponin foams

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since they are very stable even at low concentrations, like 0.01 %. With the parameter sta-

bility of the maximum foam height tfmax-5% different collapse profiles can be quantified. But

as well as for the foam half-life time not all foams collapse by 5 %. A limit of less than 5 %

was tested but yielded for tfmax-1% CVs of 24 % and 41 % and tfmax-3% CVs of 47 % and 22 %

for concentrations of 0.01 and 0.003 % saponin, respectively. Because of the high varia-

tions and limitations for the required amount of foam decay this parameter is not further

discussed in this work. The results for the relative drainage dn% had high variations and

therefore uncertainties. Even in higher density foams (0.01 % saponin) the variations were

well above 10 %. These variations can be explained, especially in lower density foams,

because the change in the fluid levels is very small for each of the analyzed saponin con-

centrations and at maximum only 10 mm. In this range even small measurement errors have

a high impact on the relative drainage and high variations occur.

Very similar to the high uncertainties in the relative drainage, the variations in foam density

fden,n arise from the dependency on the liquid height. That is why CVs are similar between

fden,n and dn%. The foam density is not an appropriate parameter to reliably quantify foam

structure. For this reason a new method using the differences in brightness of the foam

profiles was developed, see Figure A-3. From the brightness distribution the two parame-

ters BDm,n and BDw,n were derived. As in Table A-4 displayed, the variation using the new

method are clearly below the variations using fden,n. For BDm,n and BDw,n the variations of

the higher density foam were smaller and beneath 5 %. For the lower density foams the

median BDm,n is around 5 % but the variation of the width BDw,n is at 12 %. Although 12

% is still a high variation, this value is considerably lower compared to the foam density

values.

To summarize, for all further analysis kf, fn%, fden,n, BDm,n, BDw,n and foam picture were used

to characterize foaming, foam stability and foam structure.

Curriculum Vitae

187

Sandra Böttcher Technische Universität Berlin

Curriculum Vitae

Sandra Böttcher

Education

Oct 13-Jan 17 PhD (Dr. Ing.)

Prof. Dr. Stephan Drusch

Food Technology and Food Material Science

Technische Universität Berlin

Dissertation:

Saponins as natural foaming and emulsifying agents

Oct 11-Sep 13 Master of Science (1.3) with distinction

Food Science and Technology

Beuth University of Applied Sciences Berlin

Master thesis:

Off-flavor masking of secondary lipid oxidation products

by pea dextrin

Project thesis:

Determination of cancerous contaminants in home-made

foods like toast and grilled meat using GC-MS

Oct 08-Sep 11 Bachelor of Science (1.3)

Food Science and Technology

Beuth University of Applied Sciences Berlin

Bachelor thesis (Symrise AG):

Extraction and crystallization of active ingredients from

the bark of ash tree

Project thesis:

Coazervation of alginates to form liquid-filled spheres

for oral consumption

July 08 A-levels (1.5)

Johann-Wolgang-von-Goethe Gymnasium Chemnitz

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Curriculum Vitae

Sandra Böttcher Technische Universität Berlin

Awards and scholarships

Oct 13-Dez 16 Graduate scholarship of Friedrich Naumann Foundation

for Freedom

Oct 09-Sep 13 Student scholarship of Friedrich Naumann Foundation

for Freedom

Mar 15 Fellowship award of Harvard School of Public Health

International experience

Jun-Jul 15 Research visit, Free University Bolzano

Research group of Prof. Dr. Matteo Scampicchio

Characterization of saponin/beta-lactoglobulin-interactions

with isothermal titration calorimetry

Sep-Oct 07 Student exchange, Fargo, North Dakota

German American Partnership program (GAPP)

Fargo North High School

Professional Experience

Okt 13-Jan 17 Teaching assistant, Technische Universität Berlin

Lectures on design of experiments, molecular inclusion

Supervision of several bachelor/master theses

Organization of practical lab workshops for students

Apr 10-Apr 13 Teaching assistant, Beuth University of Applied Sciences

Process Engineering, Food Chemistry

Feb-Aug 11 Symrise AG, Holzminden

Internship and bachelor thesis at Competence Center for Health

& Nutrition

Aug-Sep 09 Deutsche Extrakt Kaffee GmbH, Berlin

Internship in Quality Management and Research

Feb-Mar 09 Ehrmann AG, Freiberg

Internship in Research and Development

Jul-Sep 08 Ehrmann AG, Freiberg

Internship in Research and Development