Unique Emulsions based on recombinant Hydrophobins

Unique Emulsions based on ecombinan Hyd ophobins Disse a ion zu E langung des Dok o g ades de Fakul ä ü Biologie, Chemie und Geowissenscha en an de Uni e si ä Bay eu h Vo geleg on Diplom-Biochemike Ma in He ibe Rege Immen eu h im Dezembe 2011 2 Die o liegende A bei wu de on Ap il 2009 bis Ok obe 2011 un e Lei ung on P o . D . em. Heinz Ho mann ange e ig . Volls ändige Abd uck de on de Fakul ä ü Biologie, Chemie und Geowissenscha en de Uni e si ä Bay eu h genehmig en Disse a ion zu E langung des akademischen G ades eines Dok o s de Na u wissenscha en (D . e . na .) P omo ionsgesuch einge eich am: 7. Dezembe 2011 Tag des wissenscha lichen Kolloquiums: 25. Ap il 2012 P ü ungsausschuss: P o . D . em. Heinz Ho mann (E s e Gu ach e ) P o . D . S ephan Fö s e (Zwei e Gu ach e ) P o . D . Axel Mülle (Vo si zende ) P o . D . Thomas Scheibel 3 „Ideen, wie absolu e Gewisshei , absolu e Genauigkei , endgül ige Wah hei und so o , sind E indungen de Einbildung und haben in de Wissenscha nich s zu suchen...“ Max Bo n „Am Ende gil doch nu , was wi ge an und geleb – und nich was wi e sehn haben“ A hu Schni zle 4 Table o Con en s Zusammen assung......................................................................................................................5 Summa y ....................................................................................................................................6 1 In oduc ion.............................................................................................................................7 1.1 Hyd ophobins...................................................................................................................7 1.2 Gene al Rema ks on Emulsions.....................................................................................11 2 Mo i a ion.............................................................................................................................18 3 Synopsis ................................................................................................................................19 3.1 Physicochemical cha ac e iza ion o H S a P o eins ® (Publica ion A).......................19 3.2 H S a P o eins ® as emulsi ie s (Publica ion A)...........................................................22 3.3 H S a P o ein ® B in combina ion wi h solids (Publica ion B & C) ............................25 3.4 Replacemen o H S a P o eins ® o o he amphiphiles ...............................................31 3.4.1 P o eins (Publica ion D)..........................................................................................31 3.4.2 Polyme s and Su ac an s (Publica ion E)...............................................................33 3.5 Pe spec i es....................................................................................................................36 4 Abb e ia ions and Symbols ..................................................................................................37 5 Re e ences.............................................................................................................................38 6 Lis o publica ions................................................................................................................43 Publica ion A........................................................................................................................44 Publica ion B........................................................................................................................55 Publica ion C........................................................................................................................66 Publica ion D........................................................................................................................77 Publica ion E........................................................................................................................99 Publica ion F ......................................................................................................................109 7 P esen a ions a in e na ional mee ings...............................................................................111 8 Danksagung.........................................................................................................................112 9 E klä ung.............................................................................................................................113 5 Zusammen assung Hyd ophobine sind Pilzp o eine aus e wa 100 Aminosäu en. Hyd ophobine sind die am s ä ks en obe lächenak i en, na ü lichen P o eine mi ausgesp ochene Tendenz zu Selbs agg ega ion. Au g und ih e ielsei igen Eigenscha en wi ken Hyd ophobine in ielen S uk u en des Pilzes, un e ande em als Man elsubs anz de Hyphen. Anwendungen de Hyd ophobine sind im g oßen Maßs ab bishe an den Kos en und dem Au wand de na ü lichen Hyd ophobin Reinigung geschei e . Dies ände e sich g undlegend du ch den Einsa z weiße Bio echnologie g undlegend, welche ekombinan e Hyd ophobine heu e im g oßen Maßs ab zugänglich mach . In diese A bei we den zwei ekombinan e Hyd ophobine beispielha ü diese P o einklasse au Ih e Fähigkei zu Emulsionss abilisie ung hin un e such : H S a P o ein ® A und B. Die physikalisch-chemische Cha ak e isie ung de Hyd ophobine zeig , dass sich diese ekombinan en P o eine in ih e Obe lächenak i i ä a sächlich wie die na ü lichen Hyd ophobine e hal en. De Einsa z ekombinan e Hyd ophobine als Emulga o üh zu Ausbildung gela ige Öl- in-Wasse Emulsionen, die sich nich meh au ennen, also wede Au ahmen noch Koaleszenz zeigen. Die Emulsionen we den soga zei abhängig, in S unden ode Tagen, noch wesen lich s abile . De G und is de kine isch kon ollie e Au bau eines äumlichen Ne zwe kes aus Hyd ophobin, das die Öl öp chen umgib . Diese A de Emulsionss abilisie ung is neua ig au dem Feld de Emulsions echnologie. Es wu de ge unden, dass Hyd ophobine in de Lage sind, an Clays, also scheibchena ige Schich silika pa ikel, eilweise ode olls ändig zu adso bie en. Hie aus e gib sich die Möglichkei , die Eigenscha en de Sys eme Wasse , Öl und Schich silika kon ollie zu e ände n. Es wu de gezeig , wie du ch syne gis ische E ek e homogene und höchs s abile Picke ing Emulsionen he ges ell we den können, die sich du ch hohen Ölgehal bei ex em ge ingem Emulga o an eil auszeichnen. Die plana en Schich silika e können auch du ch s äbchen ö mige Teilchen e se z we den. Boehmi enadeln bilden eben alls in Ve bindung mi Hyd ophobin Picke ing Emulsionen. Abschließend wi d gezeig , wie Hyd ophobin in de Kombina ion mi Schich silika auch du ch ande e P o eine, Amphiphile ode Tenside e se z we den kann. Du ch Eins ellen de P äpa a ionsbedingungen, de Emulga o enkonzen a ion ode des Ölmassenb uchs besi z man nun ein neues, uni e selles We kzeug, um Picke ing Emulsionen mi gewünsch en Eigenscha en he zus ellen. 6 Summa y Hyd ophobins a e e y in e es ing p o eins o ungal o igin. Beside hei ela i ely small size o a ound 100 amino acids, hey a e well known o be he mos su ace ac i e, na u al p o eins ha ha e a s ong endency o sel -assembly. Due o hei e sa ile p ope ies hyd ophobins a e p esen in di e en ungal s uc u es, like as coa e s o hyphae. These di e si ied p ope ies o hyd ophobins aised g ea in e es among scien is s. Possible applica ions in su ace modi ica ion o emulsion indus y we e always es ic ed by he cos and e o o na u al hyd ophobin pu i ica ion. This changed d ama ically by he use o whi e bio echnology esul ing in he a ailabili y o high amoun s o ecombinan hyd ophobins nowadays. This s udy s a ed wi h he physicochemical cha ac e iza ion o wo ecombinan hyd ophobins, called H S a P o eins ® A and B. Bo h show a ema kable, ime-dependen su ace ac i i y as well as a dis inc agg ega ion beha iou indica ing hem o ha e he ypical p ope ies o na u al hyd ophobins. The use o he ecombinan hyd ophobins as emulsi ie esul ed in he o ma ion o gel-like oil in wa e emulsions. In e es ingly, wi hou he occu ence o ypical emulsion ins abili y p ocesses like c eaming o coalescence, hese emulsions showed signi ican aging e ec s. We conclude hem o be he consequence o he ime-dependen o ma ion and p og ession o a sel -suppo ing, h ee-dimensional p o ein ne wo k ha e ol es in he emulsion. The sel -assemble endency o ecombinan hyd ophobins is clea ly no limi ed by adso p ion o he oil-wa e in e ace. Ob iously he long e m s abili y o he emulsion is de e mined by he s icky cha ac e o he hyd ophobin coa ed oil d ople s ha a ac each o he in he sho ange dis ance. This ype o emulsion s abiliza ion mechanism is absolu ely no el in he ield o emulsion echnology. Mo eo e we used he hyd ophobins’ abili y o su ace modi ica ion in o de o coa disk-like clay pa icles. These clay-hyd ophobin sandwiches we e used o he o ma ion o Picke ing Emulsions. I u ned ou ha he syne gis ic use o clay and hyd ophobin esul ed in homogenous, long- e m s able and oo h-pas e like emulsions. The clay pa icles imp o ed s ikingly he igidi y and elas ici y o he sel -suppo ing hyd ophobin ne wo k. Subs i u ion o he clay pa icles by boehmi e needles esul ed in simila Picke ing emulsions. Finally, we epo ha i is possible o eplace hyd ophobin in combina ion wi h clay by o he p o eins, amphiphiles o su ac an s. By adjus ing he p epa a ion condi ions, he emulsi ie concen a ion o he oil mass ac ion one has a e sa ile ool o ob ain Picke ing emulsions wi h he desi ed p ope ies. A new s abiliza ion mechanism in emulsion science is in oduced, suppo ed and con i med by ou esul s. 7 1 In oduc ion 1.1 Hyd ophobins Fungi play an impo an ole in he diges ion o dead o ganic le o e s like lea es, woods o dead insec s. Fo his pu pose i is ob ious ha a huge amoun o me abolizing enzymes, like cellulases, ha e o be p oduced and sec e ed by ungi. A he same ime i is indispensable ha ungi can e ec i ely in il a e he ma e ial o be ecycled. The e o e he ungi o m apically g owing hyphae wi hin he subs a e [1]. By b anching o he hyphae a wide mycelium can be es ablished. Colonies o he ungus A milla ia bulbosa o example a e known o o m a se e al hec a es big mycelium [2]. In o de o ul ill ep oduc ion pu poses conidiopho es ha a e de i ed om ae ial hyphae a e buil up. A all s ages o ungal li e cycle one special p o ein g oup, called hyd ophobins, is in ol ed. Hyd ophobins ha e been iden i ied o be pa o he ae ial hyphae and ui bodies [3] and consequen ly as modules o he spo e wall [4]. In o de o achie e an e icien di use o he ungal ep oduc ion s uc u es hei wa e -based dispe sal is media ed by hyd ophobins [5]. E en ungal a achmen o hyd ophobic su aces is ealized by hyd ophobins [6]. Recen ly i was shown ha hyd ophobins also mask he ecogni ion o ai bo ne ungal spo es by he immune sys em [7]. Hyd ophobins he e o e seem o ake o e he unc ions o su ac an s in biological sys ems. The ques ion ha immedia ely a ises is wha special ea u es make his class o p o eins so mul i alen and unique? Hyd ophobins a e ela i ely small p o eins o abou 100 amino acids and we e i s disco e ed in he ungi Schizophyllum commune in he ea ly eigh ies o he las cen u y [8]. S udies abou hei amino acid composi ion showed ha hyd ophobins cha ac e is ically con ain a ema kable amoun o hyd ophobic amino acids as well as eigh cys eine esidues [9]. An ea ly amino acid sequence compa ison o di e en hyd ophobins led o he classi ica ion o class I and class II hyd ophobins [10]. Mo eo e bo h ypes di e in he solubili y o hei agg ega es, he so called odle s [11]. Class I hyd ophobin odle s a e much easie o dissol e han hose o med by class II hyd ophobins. Sol en s like i luo ace ic acid a e needed in o de o dissol e hem [12]. In e es ingly, beside he ob ious di e ences in he hyd opa hy pa e ns and he consequen solubili y beha iou , he eigh cys eine esidues o all hyd ophobins a e aligned in he same, symme ic way. Only he second and hi d as well as he six h and se en h cys eine esidue ack each o he di ec ly in he amino acid sequence. The emaining cys eines a e much mo e delocalized om each o he and he e o e mo e isola ed acco ding o he p ima y p o ein 8 s uc u e. The consensus sequence o he conse ed cys eine pa e n is schema ically shown in ig 1.1. X 2-38 - C -X 5-9 - C-C -X 11-44 - C -X 8-23 - C -X 5-9 - C-C -X 6-18 - C -X 2-14 Fig. 1.1 Consensus amino acid sequence o he p ese ed cys eine pa e n. Cys eine esidues a e abb e ia ed as C and a e sepa a ed om each o he by a a iable numbe o amino acids indica ed by X. Only he cys eine esidues wo and h ee as well as six and se en a e pai ed. Figu e modi ied om [13]. Mo e cla i y owa ds unde s anding i s amphiphilic unc ion was supplied by he i s a omic esolu ion s uc u e o a hyd ophobin published in 2004. The c ys al s uc u e o he hyd ophobin HFBII om T ichode ma eesei was esol ed wi h 1.0 Å [14]. The schema ic opology (A) as well as he s uc u e (B) a e shown in ig. 1.2. Fig. 1.2 Topology (A) and s uc u e (B) o he hyd ophobin HFBII o T ichode ma eesei. Cha ac e is ic ea u es o he single domain p o ein a e he ou β-shee s (indica ed as β1-4) o ming a ba el-like s uc u e as well as one α-helix (α1). The opology s uc u e was modi ied om e . [14], whe eas he e ia y s uc u e was d awn wi h he so wa e JMol; pdb numbe o HFBII: 2B97. The single domain hyd ophobin is o globula shape wi h a diame e o a ound 3 nm. The ou β-shee s a e o ien a ed an ipa allel o each o he o ming a ba el-like s uc u e. A deepe in es iga ion o he e ia y s uc u e o HFB II also poin ed ou ha he igidi y o he globula hyd ophobins is p o ided by he exis ence o ou disul ide b idges. Due o he p o ein olding e en he locally dis inc cys eine esidues ( ig. 1.1) come close o each o he in 9 he h ee-dimensional s a e and a e now able o o m chemical bonds, espec i ely disulphide b idges. These make he hyd ophobins ex emely esis an agains he mal hea ing. No sign o dena u a ion is obse ed a e incuba ion o 15 min a 90°C [15]. Beside he conse ed cys eine esidues he p ima y sequence compa ison de ec ed also se e al p ese ed hyd ophobic amino acids, like aline o leucine esidues. In he e ia y p o ein s uc u e, hey buil up a cha ac e is ic, la hyd ophobic su ace pa ch ha makes a leas 12% o he o al p o ein su ace a ea [14]. Due o hese unique p o ein s uc u al ea u es, i is ob ious ha hyd ophobins can be conside ed as igid bio-su ac an s as hey p o ide dis inc hyd ophobic and hyd ophilic p ope ies ( ig. 1.3). Fig. 1.3 Illus a ion o he posi ion o he hyd ophobic su ace pa ch in he e ia y s uc u e o HFBII om T ichode ma eesei (A). The amino acid esidues ha a e pa o he hyd ophobic pa ch a e shown in yellow. An abs ac igu e (B) was d awn in o de o poin ou mo e clea ly he dis inc amphiphilic cha ac e o hyd ophobin. The hyd ophobic pa ch is d awn in black. The e ia y s uc u e (A) was aken om e . [14]. Simple su ac an s, like Ce yl ime hylammoniumb omide (CTAB), consis o a hyd ophilic head g oup and a lipophilic ail. Due o hei amphiphilic na u e su ac an s a e su ace ac i e and lowe he su ace ension o wa e . Keeping in mind he desc ibed, exclusi e a chi ec u e o hyd ophobins, i is e iden ha e en hey we e disco e ed ea ly on as su ace ac i e agen s. In li e a u e hyd ophobins a e e e ed o be he mos su ace ac i e p o eins [16]. Fu he mo e hei s ong endency o sel -assembly [17], e en a in e aces [18], is cha ac e is ic o hyd ophobins. 16 Many p o eins, such as milk p o eins, ß-caseins o bo ine se um albumins (BSA), a e known o many decades as emulsi ie s and eno mous esea ch wo k abou he p o ein p ope ies unde di e en condi ions using a b oad ange o me hods is s ill ca ied ou . Nowadays he cus ome demands he eplacemen o su ac an s o mo e na u al emulsi ie s. The e o e p o eins a e sui able candida es. Un o una ely a lo o emulsions based on p o eins o e echnical disad an ages, like he need o high p o ein concen a ions, a as phase sepa a ion o emulsions o an ul a-sensi i i y agains ex insic ac o s, like pH, empe a u e o sal . In summa y, he pe ec p o ein emulsi ie should ul ill besides a p onounced cos ume accep ance ollowing equi emen s: - s abilize homogenous emulsions a low concen a ions - p o iding an elec os a ic and mechanical ba ie - migh in oduce an inc eased iscosi y in he emulsion phase - a ailable in su icien amoun s and cons an quali y - ole an agains s o age e ec s, like empe a u e luc ua ion Solid pa icles, which a e adso bed a he d ople , a e able o p o ide a o m o s abilizing emulsions which is known as Picke ing emulsions [39]. They ha e al eady been disco e ed 1903 by Ramsden [40]. In he las hund ed yea s a b oad ange o solid ma e ials has been in es iga ed owa ds i s s abilizing p ope ies. Me al oxides, like TiO 2 o ZnO, silica o clays a e jus a sho selec ion o he used solid pa icles [41-44]. The emulsion s abiliza ion mechanism o hese solid pa icles is almos iden ical wi h he one o amphiphilic molecules. O special in e es a e such Picke ing emulsions wi h clays. Na u al o syn he ic clays a e well s uc u ed, colloidal building blocks [45]. The disc-like clay pa icles ha e a hickness o only 1 nm. The nega i e excess cha ge is a consequence o special ion-subs i u ions, like Si by Al, Al by Mg and Mg by Li. Alkali-me al ions usually sepa a e he clay building blocks. Solu ions o ex olia ed clays a e low iscous and anspa en . In e es ingly, solu ions o clay unde go an ab up sol-gel ansi ion wi h inc easing clay concen a ion [46]. Due o hei la ge su aces o 1000 m 2 /g clays a e pe ec adso p ion subs a es o he immobiliza ion o dyes, mul i alen ca ions o su ac an s [47]. 17 The ad an ages o using clay pa icles as emulsion s abilizing agen s we e summa ized by Lagaly e al. [48]: - ela i ely small size - ac ions o di e en pa icle size a ailable - able o inc ease he iscosi y o he con inuous phase - clay su ace can easily be modi ied by adso p ion The possibili y o su ac an s o adso b on clay pa icles can also be used o p epa ing Picke ing emulsions. Such emulsions om clay-non-ionic su ac an s sys ems ha e al eady been p o en o be qui e s able [49]. Ne e heless, i seems ha nobody ied o eplace su ac an s by p o eins in combina ion wi h clays. Fu he mo e i is well known, ha hyd ophobin adso bs o hyd ophilic su aces. In his hesis, he e o e, i will be shown, ha clay-hyd ophobin sys ems a e ideal colloidal s abilize s o he o ma ion o long- e m s able, homogenous and gel-like Picke ing emulsions. Mo eo e o he combina ions o clay-p o ein as well as he eplacemen o clay o alumina powde , called boehmi e, will be in oduced and cha ac e ized o hei po en ial as s abilize o Picke ing emulsions. 18 2 Mo i a ion Despi e he echnological conside a ions and impo ance o any po en ial emulsi ie molecule, he e has been de eloping an inc easing consume in e es and p essu e o use mo e non- oxic and biodeg adable ing edien s o o mula ions, like emulsions. E en cosme ic p oduc s con ain nowadays mo e and mo e na u al ing edien s. As such p oduc s ha e a e y high consume accep ance and demand, companies consequen ly ha e a majo in e es o p oduce and es ablish inno a i e p oduc s con aining na u al compounds. The aim o my PhD- hesis was o s udy he physicochemical p ope ies o sui able, new and la ge-scale a ailable emulsi ie s wi h low oxici y, no skin i i a ion and enhanced biodeg adabili y o cosme ic use. O cou se, p o eins a e well sui ed o ul ill he men ioned equi emen s. Recen ly de eloped, in kilog am scale a ailable ecombinan ly p oduced hyd ophobins om ungal analogous, called H S a P o ein ® A and B, a oused ou in e es . The i s pa o my PhD- hesis is abou he physicochemical cha ac e iza ion o he newly de eloped H S a P o eins ®. Thus, he basic analysis included among o he s he su ace and in e ace beha io as well as hei endency o sel -agg ega ion and ilm o ma ion. As he ecombinan hyd ophobins should be used in cosme ics, he second pa concen a es on he emulsion pe o mance o he H S a P o eins ®. O special in e es is he de e mina ion o he minimum needed emulsi ie concen a ion ha is equi ed o ob ain homogenous emulsions. Mo eo e he heological p ope ies o he emulsion a e discussed. Picke ing emulsions a e well known o many decades. Howe e expe imen s abou p o eins in combina ion wi h clay ac ing as emulsi ying agen ha e no been ca ied ou a all. As hyd ophobins a e well known o adso b o hyd ophilic su aces, like clays o boehmi e pa icles, he esul ing hyd ophobin coa ed pa icles should be es ed owa ds hei emulsi ying pe o mance. Finally, my PhD hesis closes wi h he sec ion abou he eplacemen o hyd ophobin in combina ion wi h clay o o he p o eins, polyme s o su ac an s. The e ec s on emulsion s abili y, a e age d ople s size and heological p ope ies will be discussed. 19 3 Synopsis 3.1 Physicochemical cha ac e iza ion o H S a P o eins ® (Publica ion A) The bio echnically p oduced hyd ophobins, called H S a P o eins ®, we e ecei ed wi hou any u he a ailable in o ma ion abou hei p ope ies. The e o e a basic physico-chemical cha ac e iza ion seemed o be indispensable. H S a P o eins ® A and B, om now abb e ia ed as HPA (46 kDa; IEP: 6.15) and HPB (19 kDa, IEP: 6.15) a e soluble o a concen a ion o 5 w %. The pH was de e mined o 7.95 (HPA), espec i ely 7.54 (HPB) o 1 w % solu ions o each p o ein. Bo h bio echnical hyd ophobins ha e been es ed owa ds hei su ace and in e ace ac i i y using he d op olume echnique. Bo h alues dec ease cons an ly wi h inc easing p o ein concen a ion up o hei solubili y limi (Publica ion A, ig. 1). Compa a i e s udy o he su ace ension p o iles o i e di e en p o eins con i med he hesis ha hyd ophobins a e among he mos su ace ac i e p o eins [16]. Hyd ophobin HPB was in compa ison o i e o he p o eins, like BSA o soy p o ein, he mos su ace ac i e one (Publica ion D, ig. 1). A e y impo an p ope y o he bio echnically hyd ophobins is obse ed by moni o ing hei ime-dependen su ace ension beha io ( ig. 3.1). Fig. 3.1 Time-dependen su ace ension p o ile o HPB. Plo ed a e he su ace ension γ o e y sho d op o ma ion imes ( illed symbols: 1s/µl) as well as o e y long d op o ma ion imes (open symbols: 43s/µl). Modi ied om Publica ion A, ig. 2. Applying a longe d op o ma ion ime ob iously leads o a dec eased su ace ension o HPB ( ig. 3.1). In his wo k, i is also shown ha he dec ease o he su ace ension is a esul o he o ma ion o a hin hyd ophobin ilm in he su ace laye [17]. As he same ime- 20 dependen e ec s ha e been obse ed o HPA (Publica ion A, ig. 2), he ecombinan ly H S a P o eins ® ob iously o e he same p ope ies as na u al hyd ophobins, espec i ely a dis inc su ace ac i i y as well as a endency o in e ace sel -assembly [18]. These expe imen al esul s a e no sel -e iden a all. Despi e he ac ha he usion pa ne in he case o HPA is mo e han h ee imes la ge han he ac ual hyd ophobin sequence ( ig. 1.4), he na u al hyd ophobin p ope ies a e domina ing and a e no limi ed by he usion pa ne . Ti a ion wi h HCl showed ha bo h, HPA and HPB a e nega i ely cha ged in aqueous solu ion. Tha is in ag eemen wi h he isoelec ic poin (IEP). Inc easing he HCl concen a ion in a 1% HPA solu ion leads o loccula ion a ound i s IEP and o hyd ophobin esolubilisa ion again. Mo eo e he cha ge densi y o he hyd ophobins could be de e mined by i a ion o 0.16 e/nm 2 (HPA) and 0.10 e/nm 2 (HPB). By i a ing he ecombinan ly hyd ophobins wi h HCl, hei cha ge can be uned om nega i e o e neu al o posi i e. This ansi ion is accompanied by a change in he su ace ension beha io o he hyd ophobin (Publica ion A, ig. 3). In gene al p o eins can no only be loccula ed by achie ing hei IEP due o acid-base i a ion, bu also by in e ac ion wi h su ac an s [50] and ions [51]. T ea ing 1% HPB solu ions wi h he ca ionic su ac an CTAB and he di alen ion Ca 2+ led in bo h cases o loccula ion (Publica ion A, ig. 4). In con as o CTAB and pH ea men , adding excess Ca 2+ induced no esolubilisa ion o he hyd ophobin. By using C yo-TEM he size o he hyd ophobins was esol ed as well as explici signs o memb ane agmen s we e obse ed. In ig. 3.2 he C yo-TEM mic og aph o a 0.1% HPA solu ion is shown. Fig. 3.2 C yo-TEM o a 0.1% HPA solu ion. The size o indi idual p o ein molecules is abou 5 nm. Whi e a ows indica e he memb ane agmen o ma ion buil up by assembled hyd ophobins (Publica ion A, ig. 6). 21 The C yo-TEM esul s also con i m he endency o he bio echnical hyd ophobins o sel - assembly and ilm o ma ion as i is al eady desc ibed o na u al hyd ophobins [21]. La ge hyd ophobin agg ega es ha e also been obse ed wi h he elec ic-bi e ingence echnique [52]. Using h ee di e en me hods, espec i ely ime-dependen su ace ension, C yo-TEM and elec ic bi e ingence iden i ied he bio echnical hyd ophobins o end o sel -agg ega ion and ilm- o ma ion. In e es ingly a he same ime, Lips e al. clea ed up he mechanisms o hyd ophobin sel -assembled bilaye s and he na u e o he adhesion ene gy be ween hem [19- 20]. These esul s also con i m ou conclusions. The nex chap e is abou using hese unique hyd ophobin p ope ies in emulsion science. 22 3.2 H S a P o eins ® as emulsi ie s (Publica ion A) The i s s ep in cha ac e iza ion he H S a P o eins ® owa ds hei emulsi ying pe o mance was a compa ison wi h simple su ac an s, like CTAB. The oil mass a io was kep a 20 w %. Bo h ype o emulsi ie s o m o/w emulsions as de e mined by conduc i i y measu emen s. All samples phase sepa a ed, wi h an uppe emulsion laye and a lowe aqueous phase. In e es ingly he heology o he emulsion phases s abilized by hyd ophobins indica ed hem o be weak gels, whe eas he su ac an based ones we e iscous (Publica ion A, ig. 7). In o de o use he emulsions o cosme ic o mula ions, he nex objec i e was o ob ain homogenous emulsions. The e o e he equi ed minimum amoun o oil and hyd ophobin had o be de e mined. I u ned ou , ha wi h inc easing he oil mass ac ion Φ o mo e han 0.65, homogenous emulsions could al eady be ob ained wi h li le as 0.02 w % HPB (Publica ion A, ig. 15). O special echnical in e es was he obse a ion, ha he use o bio echnical hyd ophobins as emulsi ying agen s is no accompanied wi h he es ic ion o special oil ypes. Mo eo e a b oad ange o oils, s a ing om he apola dodecane o e he silicone oil polydime hylsiloxane (PDMS) o he pola oil oc ylme hoxycinnama e (OMC) could easily be emulsi ied. An exempla y heog am o a homogenous emulsion con aining 1% HPB and an oil mass ac ion Φ o 0.65 dodecane is shown in ig. 3.3. Fig. 3.3 Rheog am (τ=0.5 Pa) o he emulsion con aining 1% HPB and an oil mass ac ion Φ o 0.65 dodecane measu ed di ec ly a e p epa a ion. The emulsion was p epa ed a a shea a e o 9000 pm. Blue: S o age modulus G’ [Pa], ed: Loss modulus G’’ [Pa] and g een: Viscosi y η [Pas]. Da a used in Publica ion A, ig. 12. The heog am shown in ig. 3.3 ob iously indica es he emulsion o be gel-like. Bo h, he s o age modulus G’ as well as he loss modulus G’’ a e equency independen . The iscosi y dec eases linea ly. 23 While he emulsions p epa ed unde hese condi ions did no show any ins abili y mechanisms, he elas ic p ope ies inc eased wi h u he incuba ion a oom empe a u e. A dis inc ipening o he emulsion laye was obse ed, he s o age modulus G’ inc eased almos h ee imes wi hin se en days (Publica ion A, ig. 13). This e ec can be explained by he ime- dependen e olu ion o he h ee-dimensional hyd ophobin ne wo k ha o ms a ound he emulsion d ople s. The hyd ophobin molecules do no loose hei abili y o sel -assembly due o in e ace adso p ion. As indica ed al eady by ime-dependen su ace ension measu emen s ( ig. 3.1), he hyd ophobins unde go con o ma ional ea angemen s a he in e ace ollowed by pa ial en anglemen o he adso bed hyd ophobin molecules. The ne wo k s i ens wi h ime as p o en by he inc ease o he s o age modulus. The emulsion s abili y mechanism p o ided by hyd ophobin is he e o e qui e unique and new. Ob iously he hyd ophobin coa ed emulsion d ople s a ac each o he in he sho ange [20]. Ac ually a ac ion o emulsion d ople s p omo es ypical emulsion ins abili y mechanisms, like coalescence. This is e iden ly no he case he e. A hyd ophobin ma ix e ol es wi hou changing he isual appea ance o he emulsion. An addi ional expe imen was pe o med in o de o con i m he desc ibed ne wo k o ma ion. By long e m cabine d ying o an emulsion based on hyd ophobin, he oil and wa e was emo ed. A e wa ds REM-mic oscopy o he ob ained ligh ma e ial was pe o med ( ig. 3.4). Fig. 3.4 REM mic og aph o he d ying esidue o an emulsion con aining 1% HPB and an oil mass ac ion Φ o 0.65 dodecane. The ba ep esen s 10 µm (Publica ion A, ig. 14). Fig. 3.4 eminds o a sponge-like, h ee-dimensional s uc u e. The hole size is iden ical wi h he de e mined emulsion d ople size be o e s a ing he d ying p ocess (Publica ion A, ab. 1). 24 An addi ional indica ion o a p ocessed emulsion d ople agg ega ion was ob ained by using compu e omog aphy (CT). Agg ega es wi h a ypical size o 200 µm we e esol ed (Publica ion A, ig. 9). Emulsions based on he H S a P o eins ® a e gel-like and ha e a yield-s ess due o he exis ence o hyd ophobin coa ed oil d ople s. These s icky pa icles o m a h ee-dimensional ne wo k wi hin he emulsion. Finally he dilu ion o homogenous emulsions wi h wa e showed, ha he emulsion phases con ac s again. Ob iously he emulsion d ople s a ac each o he . Ob aining homogenous emulsions wi h less han 65 w % oil could be ealized by in oducing a yield s ess in o he emulsions. Fo a be e scien i ic unde s anding as well as o indus ial p ocessing, he in luence o ex insic ac o s like hea ing, adding glyce ole o applying di e en shea a es while homogeniza ion is o big in e es . Hea ing a eshly p epa ed emulsion did nei he change he isual appea ance no he emulsion d ople size, bu led o a doubling o he s o age modulus G’ (Publica ion A, ig. 11). This e ec can be explained by he accele a ed s i ening o he hyd ophobin ilm in he emulsion ma ix. Consequen ly no aging e ec s could be de ec ed o he hea ed emulsion. Mo eo e i can be concluded ha hyd ophobins emulsi ying pe o mance is no a ec ed by hea ing, he emulsion emained in i s homogenous s a e. The emulsions p epa ed om hyd ophobin and dodecane had a whi e isual appea ance. Ma ching he e ac i e index by adding inc easing amoun s o glyce ole is a well known me hod in o de o ge samples anspa en [53]. A ce ain imp o emen o he emulsions anspa ency was ob ained by adding 40-60 w % glyce ole, howe e he samples did no ge comple ely anspa en . This e ec migh also be ela ed o he ne wo k o ma ion, as big agg ega es will sca e he ligh mo e e ec i ely. By applying a su icien shea a e, he emulsion d ople size achie es alues ha a e nea o heo e ical ones de e mined wi h he co e shell model (Publica ion A, ab. 2, equa ion 2). Fu he mo e a dec eased emulsion d ople size is ela ed o an inc eased elas ici y o he hyd ophobin ne wo k (Publica ion A, ig. 12). A smalle a e age d ople size achie ed a a cons an oil mass a io esul s in a highe amoun o d ople s. These d ople s will ha e mo e connec ions and in e ac ions wi h each o he , wha consequen ly esul s in highe s o age moduli. In summa y, emulsions p epa ed om he H S a P o eins ® p o ide long- e m emulsion s abili y by he o ma ion o a h ee-dimensional ne wo k wi h he emulsion d ople s apped inside. As he HPB is mo e ela ed o na u al hyd ophobins, we exclusi ely used i in he ollowing s udies. 25 3.3 H S a P o ein ® B in combina ion wi h solids (Publica ion B & C) Clays a e used in cosme ics as heological addi i es. In combina ion wi h non-ionic su ac an hey ha e been used in o de o p epa e Picke ing emulsions [49]. In his s udy, he adso p ion o hyd ophobin o clay is e alua ed as well as bo h pa icles a e es ed owa ds hei abili y o s abilize Picke ing emulsions. Despi e he ac ha bo h, HPB and clay possess a nega i e excess cha ge, hey ob iously bind o each o he . Mix u es o he wo compounds a e u bid (Publica ion B, ig. 1). As al eady men ioned, hyd ophobins can bind o hyd ophilic su aces. The adso p ion o HPB o clay, espec i ely a Laponi e was e alua ed quali a i ely by C yo-TEM (Publica ion B, ig. 4) as well as quan i a i ely by su ace ension measu emen s (Publica ion B, ig. 2). 0.5 w % clay can bind h ee imes as much HPB (Publica ion B, ig. 3). This makes i concei able ha no all o he hyd ophobin is a anged in monolaye s a he wo sides o he disc-like clay pa icles, bu also in bi- o mul ilaye s [19]. The hyd ophobin coa ed clay pa icles can he e o e be assumed as s icky o amphiphilic sandwiches. Emulsions s abilized by HPB o clay alone and p epa ed wi h he high p essu e emulsi ie a 1000 ba a e inhomogeneous sys ems. Howe e , using bo h, clay and HPB syne gis ically as emulsi ying agen s esul ed in Picke ing emulsions wi h amazing oo h-pas e, gel-like p ope ies ( ig. 3.5). Fig. 3.5 Syne gis ic emulsi ying ac ion o HPB and clay esul s in oo h-pas e, homogenous and gel-like emulsions. The shown one day old emulsions con ained 0.5 w % o HPB o /and 0.5 w % clay, he oil mass ac ion Φ was ixed a 0.65 PDMS. Homogeniza ion was ca ied ou wi h he high p essu e emulsi ie (Publica ion B, modi ied g aphical abs ac ). 32 Simila ends as obse ed o he isual appea ance ha e been de ec ed in he ime-dependen beha iou o he a e age emulsion d ople size (ads) (Publica ion D, ab. 2). The ads s ayed cons an in he case o using soy p o ein and HPB, wha indica ed he emulsions o be s able agains coalescence. Simila ends we e iden i ied by e alua ing he heological p ope ies o he emulsions (Publica ion D, ig. 3). Gel-like emulsions ha unde go aging e ec s we e ob ained in he case o using soy p o ein o HPB. I has o be no ed ha he inc ease o he s o age modulus in he case o BSA was a consequence o he oil squeezing (Publica ion D, ig. 4). In conclusion he soy p o ein isola e did ha e he bes emulsi ying abili ies o all used p o eins. Acco dingly all p o eins ha e been used in combina ion wi h 0.5 w % clay in o de o s abilize Picke ing emulsions. Due o he syne gis ic use o p o ein and clay, an eno mous aise in he emulsi ica ion abili y o yeas ex ac and Plan asol W could be obse ed (Publica ion D, ig. 5). Mo eo e he emulsion p epa ed om HPB and soy p o ein in combina ion wi h clay did no longe phase sepa a e wi h inc easing ime. By aising he oil mass ac ion Φ om 0.5 o 0.65, all p o ein-clay combina ions esul ed in homogenous emulsions (Publica ion D, ig. 7). The smalles a e age d ople size could be ob ained wi h he combina ion o BSA and clay (Publica ion D, ab. 5), whe eas he mos s able, gel-like Picke ing emulsions we e p oduced om he combina ion HPB and clay ( ig. 3.10). Fig. 3.10 T end o he s o age moduli G’ (τ=0.5Pa) e alua ed in dependency o he s o age ime, espec i ely di ec ly a e p epa a ion and a e h ee days incuba ion a oom empe a u e. All Picke ing emulsions con ained 0.5 w % p o ein, 0.5 w % Laponi e XLG and an oil mass ac ion Φ o 0.65 PDMS and we e p epa ed a 300 ba (Publica ion D, ig. 8). 33 As one can conclude om ig. 3.10, i is possible o ob ain gel-like Picke ing emulsions con aining high amoun s o oil by using 0.5 w % p o ein and 0.5 w % clay. All Picke ing emulsions showed aging e ec s due o he ime-dependen s i ening o he h ee dimensional p o ein ilms. The absolu e alue o G’ is depending on he used p o ein. The mos gel-like emulsion is p epa ed om clay and yeas ex ac ( ig. 3.10, uppe ow). Howe e , he emulsion p epa ed om yeas ex ac was ex emely p one o mic obial g ow h. This is also p o en by he weakening o i s s o age moduli a e h ee days ( ig. 3.10, lowe ow). Su ely he mos s able, homogenous Picke ing emulsions can be p epa ed om he syne gis ic use o hyd ophobin and clay, bu ne e heless i is possible o eplace i by o he p o eins. By a ying he p o ein in combina ion wi h o wi hou clay and by adjus ing he oil mass ac ion Φ, one has a e y e ec i e ool o ine- une he desi ed emulsion p ope ies. 3.4.2 Polyme s and Su ac an s (Publica ion E) As he p e ious sec ion was abou eplacing hyd ophobin in combina ion wi h clay by o he p o eins, i is only logical o check he in luence o eplacing p o eins by polyme s and su ac an s. Wi h he excep ion o anionic su ac an s, like Sodium-Dodecylsul a e (SDS), all o he ypes o su ac an s o polyme s bind o clay pa icles. The concen a ion-depending adso p ion o su ac an s o clay pa icles can be moni o ed by su ace ension measu emen s. A schema ic su ace ension p o ile was ecen ly sugges ed (Publica ion E, ig. 1). E en non-ionic su ac an s bind o clays [55]. This adso p ion has o be he consequence o hyd ophobic in e ac ion be ween bo h pa icles. Consequen ly clay pa icles ha e o be somehow hyd ophobic. Ne e heless soluble clay pa icles a e no su ace ac i e. This ob ious an agonism can be explained by conside ing he low in e ace concen a ion o he clay pa icles. Consequen ly he obse ed su ace p essu e is oo low o signi ican ly in luencing he su ace ension (Publica ion E, heo e ical pa ). The used amphiphiles in his s udy we e: - ca ionic su ac an C16-T ime hylammoniumb omide (CTAB) - non-ionic su ac an Iso idecyloc ae hylenglycole he (C 13 O 8 ) - zwi e ionic su ac an Te adecyldime hylaminoxide (TDMAO) - non-ionic iblock copolyme Plu onic F38 - poly inylalcohol (PVA) 34 - poly inylpy olidone (PVP) - poly-diallyldime hylammoniumchlo ide (DADMAC) Picke ing emulsions con aining 0.5 w % amphiphile, 0.5 w % clay and an oil mass ac ion Φ o 0.5 PDMS ha e been p epa ed ( ig. 3.11). Fig. 3.11 Picke ing emulsions om di e en 1:1 mix u es o amphiphile and clay p epa ed a 300 ba . All samples con ained 0.5 w % amphiphile, 0.5 w % Laponi e XLG and 50 w % polydime hylsiloxane (PDMS) and we e pho og aphed a e 1 d (Publica ion E, ig. 2). In e es ingly all combina ions o clay and amphiphile, beside he one p epa ed om DADMAC, esul in homogenous emulsions ( ig. 3.11). Mo eo e he samples wi h CTAB, C 13 O 8 , TDMAO, HPB, PVA and PVP did no o m a smoo h, ho izon al meniscus, indica ing hei gel-like p ope ies. Especially he oo hpas e like pe o mance o he Picke ing emulsion p epa ed om PVA can e iden ly be seen. A deepe compa ison o hese Picke ing emulsions, con aining he s o age modulus G’ (τ = 0.5 Pa and = 1 Hz) and he needed shea s ess τ [Pa] o b eak he elas ic beha iou (i p esen ) o he emulsion was pe o med (Publica ion E, ab. 3). A e wo mon h incuba ion a oom empe a u e only he Picke ing emulsions based on TDMAO and Plu onic F38 did phase sepa a e, all o he Picke ing emulsions did no show any ins abili y mechanisms. The o igin o he gel-like p ope ies in he case o using hyd ophobin and clay syne gis ically ha e been discussed al eady (Chap e 3.3). In he case o o he polyme s o su ac an s i was shown ha he su ace co e age o he clay pa icles hea ily in luences he gel-like p ope ies o he co esponding Picke ing emulsions. While adding small amoun o su ac an can boos he emulsi ying p ope ies o he solu ion, high amoun s dec ease hem apidly (Publica ion E, ig. 5 and 6). 35 In his sec ion i was shown ha almos all used amphiphilic compounds bind on nega i ely cha ged clay pla ele s. S able Picke ing emulsions can be p epa ed om samples wi h 50 % wa e and 50 % oil wi h as li le as 0.5 % clay and low amoun s o amphiphile. The Picke ing emulsions a e o he o/w- ype and ha e gel-like p ope ies. The shea modulus o hese phases can be a ied be ween a ew Pascal and se e al housand Pascal o small changes in he composi ion on he clay su ace. The syne gis ic use o clay and amphiphile is a no el emulsi ying me hod ha was in oduced as a main pa o his PhD- hesis. The gel-like p ope ies we e no a consequence o he sol- gel ansi ion o he clay pa icles, as hei concen a ion was qui e a away om his poin . Mo eo e he o ma ion o a h ee-dimensional amphiphile ne wo k ha is s eng hened by he clay pa icles gua an ees he long e m s abili y o he ob ained Picke ing emulsions. 36 3.5 Pe spec i es In summa y he s abili y o Picke ing emulsions is inc eased when p o eins and clays a e used syne gis ically. So he sys ems seem o be app op ia e o a ious, di e en applica ions. Howe e p o eins a e p one o mic obial diges ion. I is shown ha his e ec can be p e en ed by he eplacemen o p o eins by su ac an s o polyme s. So a b oad a ea o applica ions in a ious ields is now opened by ou pionee wo k. 37 4 Abb e ia ions and Symbols ads a e age d ople size aec anionic exchange capaci y BSA Bo ine Se um Albumin cmc c i ical micelle concen a ion C yo-TEM c yogenic ansmission elec on mic oscopy CT compu e omog aphy CTAB ce yl ime hylammoniumb omid DADMAC poly-diallyldime hylammoniumchlo ide HPA H S a P o ein ® A HPB H S a P o ein ® B IEP isoelec ic poin OMC oc yl-me hoxycinnama e PDMS polydime hylsiloxane PVA poly inylalcohol PVP poly inylpy olidone pm e olu ions pe minu e SDS sodium-dodecylsul a e SEM scanning elec on mic oscopy TDMAO e adecyldime hylaminoxide TEM ansmission elec on mic oscopy Φ oil mass ac ion 38 5 Re e ences [1] Wessels JGH, Cell wall syn hesis in apical hyphal g ow h, In . Re . Cy ol., 1986, 104, 37-79. [2] Smi h ML, B uhn JN and Ande son JB, The ungus A milla ia bulbosa is among he la ges and oldes li ing o ganisms, Na u e, 1992, 356, 428-431. 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[32] Saigal T, Dong H, Ma yjaszewski K and Til on RD, Picke ing Emulsions S abilized by Nanopa icles wi h The mally Responsi e G a ed Polyme , Langmui , 2010, 26, 15200-15209. [33] Tigges B, Dede ichs T, Mölle M, Liu T, Rich e ing W and Weichold O, In e acial p ope ies o emulsions s abilized wi h su ac an and nonsu ac an coa ed boehmi e nanopa icles, Langmui , 2010, 26, 17913-17918. [34] S. McClellan and E. F anses, E ec o concen a ion and dena u a ion on adso p ion and su ace ension o bo ine se um albumin, Coll. & Su . B: Bioin e aces, 2003, 28, 63-75. [35] Sjoblom J, Emulsions – A undamen al and p ac ical app oach, Kluwe Academic Publishe s, 1992, Ne he lands. [36] Sengup a T, Razumo sky L and Damoda an S, Ene ge ics o P o ein−In e ace In e ac ions and I s E ec on P o ein Adso p ion, Langmui , 1999, 15, 6691-7001. [37] Mc Clemen s DJ, P o ein-s abilized emulsions, Cu . Opin. Coll. In e . Sci., 2004, 9, 305-313. [38] Van Aken GA, Blijdens ein TBJ and Ho um NE, Colloidal des abilisa ion mechanisms in p o ein-s abilized emulsions, Cu . Opin. Coll. In e . Sci., 2003, 8, 371-379. [39] Picke ing SU, CXCVI.-Emulsions, J. Chem. Soc. T ans, 1907, 91, 2001-2021. 41 [40] Ramsden W, Sepa a ion o Solids in he Su ace-laye s o Solu ions and Suspensions, P oc. R. Soc. Lond., 1903, 72, 156-164. [41] Chen T, Col e PJ and Bon SAF, O ganic-Ino ganic Hyb id hollow sphe es p epa ed om TiO 2 -S abilized Picke ing Emulsion Polyme iza ion, Ad anced Ma e ials, 2007, 19, 2286-2289. [42] He Y, P epa a ion o polyanilline/nano-ZnO composi es ia a no el Picke ing emulsion ou e, Powde Technology, 2004, 147, 59-63. [43] Tia ks F, Land es e K and An onie i M, Silica Nanopa icles as Su ac an s and Fille s o La exes made by miniemulsion polyme iza ion, Langmui , 2001, 17, 5775- 5780. [44] Ashby NP and Binks BP, Picke ing emulsions s abilised by Laponi e clay pa icles, Phys. Chem. Chem. Phys., 2000, 2, 5640-5646. [45] Sposi o G, Skippe NT, Su on R, Pa k S-H, Sope AK and G ea house JA, Su ace geochemis y o he clay mine als, P oc. Na l. Acad. Sci. USA, 1999, 96, 3358-3364. [46] Shalke ich A, S adne A, Bha SK, Mulle F and Schu enbe ge P, Clus e , glass, and gel o ma ion and iscoelas ic phase sepa a ion in aqueous clay suspensions, Langmui , 2007, 23, 3570-3580. [47] Yamaguchi Y and Ho mann H, In e ac ion be ween saponi e and ca ionic, zwi e ionic and nonionic su ac an s, Coll. & Su . A, 1997, 121, 67-80. [48] Lagaly G, Reese M and Abend S, Smec i es as colloidal s abilize s o emulsions I. P epa a ion and p ope ies o emulsions wi h smec i es and nonionic su ac an s, Applied Clay Science, 1999, 14, 83-103. [49] Lagaly G, Reese M and Abend S, Smec i es as colloidal s abilize s o emulsions II. Rheological p ope ies o smec i e-laden emulsions, Applied Clay Science, 1999, 14, 279-298. [50] Gull N, Sen P, Khan RH and Din K, In e ac ion o Bo ine (BSA), Rabbi (RSA), and Po cine (PSA) Se um Albumins wi h Ca ionic Single-Chain/Gemini Su ac an s: A Compe a i e S udy, Langmui , 2009, 25, 11686-11691. [51] Molina-Bolí a JA, Galis eo-González F and Hidalgo-Al a ez R, S abiliza ion o p o ein-la ex complexes a high ionic s eng h, Coll. & Su . B: Bioin e aces, 1996, 8, 73-80. [52] Holzheu S and Ho mann H, In luence o non-ionic adso bing subs ances on he anomalous Ke e ec o hec o i e dispe sions, P og. Coll. & Poly. Sci., 2000, 115, 265-269. binding o Ca 2+ ions. Resul s o such i a ions a e shown in Fig. 4. The binding o he ca ionic su ac an CTAB leads i s o loccula ion and hen o esolubilisa ion. In his p ocess he p o eins a e comple ely sa u a ed wi h he su ac an molecules. Du ing he i a ion o he p o eins wi h CTAB he su ace ension eaches i s a minimum and hen passes h ough a maximum. Finally he su ace ension o he pu e CTAB solu ion is eached when he ee monome solu ion o CTAB eaches he c i ical micella concen a ion (cmc) (Fig. 5). Ob iously he p o ein solu ions oam when hey a e eshly p epa ed. The oam s abili y depends e y much on he pH and he cha ging deg ee o he p o eins. In e es ingly he samples sho ly be o e and a e p o ein loccula ion ha e bes oaming p ope ies. A C yo-TEM mic og aph is shown in Fig. 6. The p o ein molecules wi h a molecula weigh o 46 kDa (HPA) and 19 kDa (HPB) a e in he size ange 5 nm) in which hey should be. Mo eo e he mic og aph shows pieces o hin ilms (ma ked wi h whi e a ows in Fig. 6) ha a e o med by in e pene a ing p o ein agg ega es. This expe imen con i ms he s ong endency o sel -agg ega ion a he ai /wa e in e ace e en o he echnical hyd ophobins as i was ecen ly obse ed by Kisko e al. o na u al hyd ophobins. 32 Mos likely hose ilms we e o med a he ai /wa e in e ace as he local concen a ion o he su ace ac i e H S a P o einscompa ed o he bulk solu ion is much highe . The ilm o ma ion could be a esul o he ime dependence o he su ace ension. We also looked o la ge molecula agg ega es wi h he elec- ic bi e ingence echnique. 39 La ge signals we e obse ed which inc eased in ampli ude and ime cons an wi h ime. These signals disappea when he hyd ophobin solu ion is il e ed h ough mic opo e il e s. Small signals appea again a e se e al days. Ob iously, he p o eins o m agg ega es wi h ime in an i e e sible p ocess. P o ein s. su ac an as emulsi ie Fou samples which we e p epa ed om aqueous solu ions o p o eins HPA and HPB, o he non-ionic su ac an C 13 E 8 , he ca ionic su ac an CTAB and 20 w % dodecane we e compa ed. All he samples a e sepa a ed in o wo phases: a lowe phase and a milky uppe phase. The olume o he uppe phase is only sligh ly la ge han ha o he pu e oil phase be o e he emulsi- ica ion p ocess. The uppe phases om he p o ein samples ha e inc eased conside ably wi h espec o he oil phase. Wi hou ha ing o he in o ma ion i can be assumed ha he uppe phases a e w/o emulsions in which a small ac ion o he aqueous phase is dispe sed in he oil phase. Howe e his is no he case, as can be concluded om conduc i i y measu emen s and he heo- logical p ope ies o he phases. In Fig. 7 heog ams o he uppe phases measu ed 1 day a e emulsi ica ion a e shown. The p o ein emulsions beha e like weak gels. The s o age modulus G0is only weakly equency dependen and is much la ge han he loss modulus G00. These a e ypical signs o a gel. The emulsions in he uppe phases ha we e p oduced wi h su ac an s can also no be w/o emulsions wi h low wa e Fig. 4 Solu ions o 1% HPB wi h inc easing amoun s o CaCl 2 and ca ionic su ac an CTAB. Excessi e p o ein loccula ion akes place a 10 mM CaCl 2 and 7 mM CTAB, espec i ely. Adding excess CTAB leads o HPB esolubilisa ion, whe eas he loccula ed s a e emains e en a highe CaCl 2 concen a ions. Fig. 5 Su ace ension gp o ile o he supe na an s o mix u es om 1% HPB and inc easing amoun o CTAB (open ci cles) in compa ison o he su ace ension o a pu e CTAB solu ion (closed ci cles). The shaded a ea indica es he CTAB concen a ion ange whe e HPB is in he loccula ed s a e. Fig. 6 C yo-TEM mic og aph o a 0.1% HPA solu ion. Whi e a ows show memb ane agmen s o med by big p o ein agg ega es. This jou nal is ªThe Royal Socie y o Chemis y 2011 So Ma e , 2011, 7, 8248–8257 | 8251 48 ac ions. In his si ua ion he iscosi ies should only be some- wha inc eased wi h espec o he iscosi y o dodecane. The iscosi ies, howe e , a e e y much inc eased and he phases show non-New onian beha iou . Fu he mo e, he emulsions ha e a conduc i i y ha is much highe han he conduc i i y an oil phase can ha e. These p ope ies, he conduc i i y and he heological p ope ies, p o e ha he emulsions mus con ain a ne wo k o an aqueous phase. I is likely ha he ne wo k is an aqueous oam ha con ains dodecane. In es iga ions o he phases wi h op ical mic oscopy indeed show ha he uppe emulsions which we e p epa ed wi h su ac an s a e high in e nal phase emulsions (HIPE). In spi e o he appea ance indica ing he phases o be w/o emulsions hey a e o/w emulsions. The oil is encased in a oam s uc u e. Such s uc u es ha e been desc ibed in he li e a u e. 40,41 The phases a e usually p epa ed in a complica ed mul i-s ep p ocess. I is he e o e su p ising ha he HIPE phases can also be o med by a simple emulsi ica ion p ocess. No all o he amphiphilic compounds a e adso bed in he ne wo k. Su ace ension measu emen s show ha some o he su ac an s a e le in he lowe aqueous phase. Ob iously no enough su ace was p oduced in he emulsi ica ion p ocess which could accommo- da e all he amphiphilic compounds on he su ace. As is ob ious om he olumes o he p o ein emulsion, hese phases con ain mo e wa e han he emulsions om he su ac- an s. I is likely he e o e ha hei s uc u e is di e en . Ligh mic og aphy o he phase p o ed hem o be no mal o/w emul- sions wi h a high polydispe si y (5–90 mm) o he oil d ople s. As i is ob ious om he gel-like beha iou o he phase, he oil d ople s wi h he adso bed p o ein ilm mus s ick oge he and o m a h ee dimensional ne wo k. All emulsion d ople s obse ed wi h ligh mic oscopy had b idging poin s wi h each o he indica ing ha hey a e uly o ming a p o ein ne wo k wi h he d ople s inco po a ed. The desc ibed esul s make i clea ha he hyd ophobin p o eins and su ac an s o m emulsions wi h di e en p ope - ies. I is likely ha his beha iou o he p o eins is due o he ac ha he su ace o he p o ein molecule keeps i s amphiphilic na u e and can o m s icky con ac s when i comes in o con ac wi h o he such su aces. P o ein–p o ein in e ac ion and en anglemen in he emulsion laye a e also suppo ed by p e ious indings. Globula p o ein molecules a he in e ace can no longe o a e eely bu a e ixed in he p o ein monolaye in a well de ined con o ma ion and aligned posi ion. 19 The molecules p obably o m a ilm in which he adso bed molecules a e connec ed wi h each o he h ough physical bonds. E idence o such ilms has been epo ed om heological measu emen s on in e acial ilms. 42 In he ollowing sec ions we s udy he p ope ies o p o ein emulsions, when pa ame e s o he sys ems a e changed. F om he desc ibed esul s and he p oposed explana ion i is al eady clea ha he heological p ope ies o he emulsions a e Fig. 7 Rheog ams o he emulsion laye s con aining 1 w % emulsi ie and F¼0.2 dodecane measu ed a s¼0.5 Pa one day a e emulsi ica ion. Blue: s o age modulus G0(Pa), ed: loss modulus G00 (Pa) and g een: iscosi y h(Pa s). 8252 | So Ma e , 2011, 7, 8248–8257 This jou nal is ªThe Royal Socie y o Chemis y 2011 49 no de e mined by he olume ac ion o he d ople s and he size dis ibu ion, bu by he p ope ies o he h ee dimensional p o ein ne wo k ha is o med in he emulsion. The in luence o glyce ol on he emulsions Many cosme ic p oduc s con ain glyce ol o di e en easons. Glyce ol lowe s he eezing poin o wa e and he samples can be exposed o lowe empe a u es wi hou losing hei homoge- nei y. Glyce ol also gi es he samples a so e ouch and keeps he wa e o longe imes. A high glyce ol concen a ion also inc eases he cmc o su ac an s. 43 E en mo e impo an o he appea ance o he samples is he ac ha glyce ol inc eases he e ac i e index o he aqueous phase 44 and can educe he e ac i e index con as be ween he wa e phase and he oil. Emulsions become he e o e mo e anspa en wi h he inc easing glyce ol con en . Glyce ol a he same ime changes he in e ac ion be ween he oil d ople s because he Hamake cons an depends on he e ac i e index o bo h he sol en and he oil and wi h he dec ease o he e ac i e index con as he a ac ion be ween he d ople s is lowe ed. This e ec has been used o p epa e s able and anspa en high in e nal phase o/w emulsions. 40 Con as ma ching o he e ac i e index can also be used in wo phase samples o L 1 /L a o inc ease he in e lamella dis ance in he L a -phase o ans o m he sys em in o a anspa en single L a -phase. 45,46 Emulsion p epa ed wi h 1% HPB p o ein and 0–60% glyce ol in he aqueous phase and oil mass ac ion F¼0.2 dodecane p o ed ha glyce ol has li le in luence on he isual appea ance o he samples up o 40% glyce ol. Howe e a s ong change in he anspa ency o he emulsion phase akes place be ween 40% and 60% glyce ol. This e ec is ob iously due o he e ac i e index ma ching. The emulsion phases do no low when he samples a e u ned upside down. In e es ingly he uppe emul- sion phase o he sample wi hou glyce ol is abou wice as la ge as he amoun o dodecane (F¼0.2) ha was used o he sample p epa a ion. The emulsion mus he e o e con ain abou equal olumes o oil and wa e . Howe e when he glyce ol concen- a ion inc eases up o 60% he olume ac ion o he emulsion laye s s ays mo e o less cons an in spi e o changing he densi y o he sol en and he Hamake cons an o he d ople in e - ac ion. Because o he Hamake cons an educ ion he a ac- ion be ween he emulsion d ople s becomes smalle and he s uc u e ac o Sshould inc ease. This has ob ious conse- quences on he s o age moduli o he emulsion phases as shown in eqn (1): G0¼nkT S(1) The s uc u e ac o Sis >1 o a ac i e pa icle in e ac ion and <1 bu >0 o epulsi e in e ac ion. 33 In his simple model in which he modulus is de e mined by he osmo ic in e ac ion o he pa icles in he sys em, he s o age modulus o dense emul- sions should be 10 6 imes smalle han he modulus o inging gels. 34 In he case o he emulsions con aining inc easing amoun s o glyce ol, he s uc u e ac o Sdec eased om a alue much la ge han 1 o smalle alues, bu s ill la ge han 1 esul ing in la ge G0 alues (Fig. 8). Compu e omog aphy o emulsions The s uc u e o emulsions can be made isible by Compu e Tomog aphy (CT). Ob iously, he con as in elec on densi y o wa e and dodecane is la ge enough o he oil s uc u es o be seen. Fig. 9 shows a mic og aph o a anspa en , homogeneous emulsion con aining 0.5% HPB and 60% glyce ol in he aqueous phase, pH 6, and an oil mass ac ion F¼0.6 dodecane. The emulsion was p epa ed wi h he o ex shake . The smalles d ople s which can be esol ed ha e a diame e o abou 50 mm. Ligh mic oscopy p o ed ha he diame e s o he oil d ople s a e in he ange o 50 mm. The mo e in e es ing in o ma ion o he CT mic og aph is, howe e , ha he small d ople s o m agg ega es wi h a ypical size o 200 mm. I is ob ious ha he size o hese clus e s is gi en by he o exing me hod. I is Fig. 8 S o age moduli G0(Pa) agains equency (Hz) measu ed a s¼ 0.05 Pa o emulsions p epa ed wi h a ious amoun s o glyce ol a e 1 day. Sample composi ion: aqueous phase: 1% HPB and 0–60% glyce ol; oil F¼0.2 dodecane. Fig. 9 Compu e omog aphy o a homogeneous p o ein emulsion. The emulsion con ained 0.5% HPB and 60% glyce ol in he aqueous phase and F¼0.6 dodecane, pH 6. The a e age d ople diame e is 50 mm. This jou nal is ªThe Royal Socie y o Chemis y 2011 So Ma e , 2011, 7, 8248–8257 | 8253 50 concei able ha hese la ge objec s o a e as whole uni s in he shea low. Emulsion wi h loccula ed p o ein In he discussion abou he p o ein solu ions i was men ioned ha he p o ein could be loccula ed by changing he pH, by adding CaCl 2 o CTAB. The h ee di e en p ocedu es ha e in common ha he ionic cha ge o he p o ein pa icles is compensa ed and he pa icles a ac each o he . We ha e used such loccula ed p o ein dispe sions o he p epa a ion o emulsions. The samples p epa ed om he loccula ed p o ein s a e using HCl and CaCl 2 look like he sample wi hou loccu- la ion agen s, bu he loccula ion wi h CTAB led o a d ama ic dec ease in he emuls ying abili y o HPB. The s o age moduli o he samples a e 1 day incuba ion a oom empe a u e a e compa ed in Fig. 10. I is in e es ing o no e ha G0 o he emulsion wi h he unmodi ied p o eins is simila o he s o age moduli o he loccula ed sys ems. I is, howe e , much la ge han he s o age modulus in he emulsion laye ha had been p oduced wi h 20 w % dodecane (Fig. 7). The excess concen a ion o p o ein in he lowe phase did ha e an in luence on he modulus o he uppe phase. I is concei able ha he wo phase sys em was a ec ed by deple ion loccula ion and ha he concen a ions o p o ein in he uppe emulsion phases and in he lowe aqueous phases we e no he same and as a consequence he s o age modulus in he 20 w % emulsion was lowe han in he single phase emulsion wi h an oil mass ac ion Fo 0.65 (Fig. 10). The mos s a ling esul is, howe e , he s o age modulus o he sample wi h added CTAB (Fig. 10). I has been no ed in he li e a u e o p o ein emulsions ha he mos s able emulsions we e ob ained wi h a loccula ed emulsi ie . 47 In he p esen sys em his is ob iously no he case, e en when only e y li le CTAB was added o compensa e he ionic cha ge o he p o ein and no as much o sa u a e he p o ein wi h a su ac an and e e se he cha ge on he p o ein. The sample wi h CTAB shows ha he uppe emulsion laye is no longe a homogeneous laye bu he emulsion has become uns able and has sepa a ed in o oil and emulsion. Ob iously coalescence be ween he d ople s has occu ed which esul ed in an excess oil phase. I is hen likely ha he small amoun o he added CTAB did no only compensa e he cha ge on he p o ein bu also e ec ed he p o ein con o ma ion. The su ac an can possibly do his by binding o he hyd ophobic pa o he p o ein molecule. By doing his he p o ein su ac an complex can no longe ac as a s icky p o ein molecule bu i ac s mo e as a no mal su ac an molecule wi h one hyd ophilic and one hyd ophobic pa . The in luence o hea ing on p o ein emulsions I is known ha he p ope ies o many p o eins a e hea sensi- i e. The bes known example is egg p o ein. Many o he p o eins a e known o loccula e when hey a e hea ed. The ansi ion o a dissol ed p o ein om he liquid s a e o he loccula ed s a e should be independen o whe he he p o ein is in he h ee dimensional bulk s a e o in he adso bed mono- molecula ilm o he emulsion. To ind ou abou he hea sensi i i y o he emulsions, we measu ed he heological p op- e ies o a eshly p epa ed emulsion and o an emulsion which was hea ea ed o a sho ime pe iod. The esul s a e shown in Fig. 11 o he emulsion con aining 0.5% HPB and F¼0.65 dodecane. The s o age modulus o he emulsion in he hea ea ed s a e is wice as high as ha o he unhea ed emulsion. This is a clea indica ion ha he s i ness o he p o ein ilm in he monolaye has become much la ge du ing he sho ime hea ea men . I is u he mo e no ewo hy ha he p ope ies o he hea ea ed emulsions no longe change wi h ime as opposed o he unhea ed emulsion. This is an indica ion ha he hea ea ed s a e o he p o ein is a e y s able s a e and can no longe change i s con igu a ion. Simila esul s wi h emulsions s abilized by p o eins, like b-casein, ha e shown ha emulsions a e usually mo e esis an o d ople agg ega ion du ing hea ing i he p o ein con igu a ion does no change comple ely upon hea ea men . 48 Shea - a e in luence on p ope ies o p o ein emulsions The emulsion d ople s in he samples a e p oduced by shea s esses ha ac on he bulk oil phases. In such si ua ions highe Fig. 10 S o age moduli G0(s¼0.5 Pa) o emulsions p epa ed om loccula ed p o ein. Final concen a ions: 0.5% HPB wi hou and wi h loccula ion agen (3.4 mM HCl, 2.5 mM CaCl 2 and 3.5 mM CTAB) and a mass a io Fo 0.65 dodecane. Fig. 11 S o age moduli G0(s¼0.5 Pa) o an emulsion wi h 0.5% HPB and F¼0.65 dodecane be o e and a e hea ing o 5 min a 92 C. 8254 | So Ma e , 2011, 7, 8248–8257 This jou nal is ªThe Royal Socie y o Chemis y 2011 51 shea s esses should p oduce smalle d ople s. Di e en shea s esses should he e o e esul in emulsions wi h d ople s o di e en dimensions and di e en p ope ies. In o de o in es iga e he in luence o shea ime on he emulsion p ope ies, he s o age modulus G0was de e mined o emulsions p epa ed wi h a cons an shea a e (5000 pm), bu di e en shea imes. The moduli we e measu ed a a small shea s ess (s¼0.05 Pa) o a oid dis up ion o he diso de ed, esh d ople s uc u e. I u ned ou ha wi h inc easing shea ime (0– 120 s) he s o age modulus G0o he emulsion was also becoming highe . Fo shea imes highe han 120 s, he co esponding emulsion modulus did no change signi ican ly any mo e. Emulsions we e p epa ed which ha e he same composi ion (1% HPB and F¼0.65 dodecane) bu ha e been emulsi ied wi h di e en mixing aids. One emulsion was p epa ed wi h a o ex shake while o he samples we e p epa ed wi h a Homo Dispe wi h e olu ions pe minu e ( pm) o 1000, 5000 and 9000 wi h a shea ime o 120 s. All samples look alike and a e homoge- neous emulsions. Howe e , hei heological p ope ies a e di e en . All samples ha e gel-like p ope ies which is e iden om he esul ha he s o age modulus is independen o equency and la ge han he loss modulus. The s o age modulus ha is he s i ness o he samples is inc easing wi h he shea s ess ha is p oduced in he echniques (Fig. 12). These esul s a e an indica ion ha he dimension o he d ople s is dec easing while he s o age moduli inc ease. This is indeed he case as i is shown in Table 1. The dimensions o he d ople s which ha e been p epa ed wi h he o ex shake a e conside ably la ge han he d ople s p epa ed wi h he high p essu e emulsi ie . Wi h an a e age d ople size o 9 mm a he highes pm s age he d ople s ha e eached a dimension which is no close o he alues ha can be calcula ed wi h he heo e ical co e shell model (eqn (2)). 3d¼R(2) whe e dis he hickness o he adso bed laye and Ris he mass a io o oil o amphiphile. F om he wo pa ame e s he adius o he emulsion d ople s can be calcula ed. I is he e o e likely ha he used emulsi ica ion de ices a e no sui ed o p oduce smalle oil d ople s in o de o comple ely use up he p o ein o he emulsion p epa a ion. The samples should s ill con ain p o eins in he aqueous phase. The aging o he emulsions wi h ime Homogeneous emulsions ha do no seem o change wi h ime can easily be p epa ed om he p o eins when he p o ein concen a ion is in he ange be ween 0.02% and 1% and he oil mass ac ion Fis la ge han 0.65. The samples did no phase sepa a e wi h ime and hei appea ance did no change. Howe e when heological measu emen s a e made a e di e en imes i u ns ou ha he elas ic p ope ies inc ease wi h ime bu app oach a cons an alue wi h ime. Fig. 13 con ains he s o age modulus wi h ime o an emulsion con- aining 1% HPB and F¼0.65 dodecane p epa ed wi h he Homo Dispe a a shea a e o 9000 pm. I is no ewo hy ha he s o age modulus mo e han doubles wi h ime. Du ing his ime he s uc u e o he emulsion as obse ed unde he mic oscope does no seem o change. I is likely he e o e ha he inc ease o he s o age modulus is gi en by he inc ease o he s i ness o he ne wo k s uc u e. In he li e a u e, pa ial en anglemen o he adso bed p o ein mole- cules is decla ed o be he eason o aging o b-casein and BSA ilms. 49 O he heolgical measu emen s showed ha no only he s o age modulus changes wi h ime, bu also he de o ma ion o he emulsion phase be o e he s o age modulus b eaks down inc eased wi h ime. This means ha he p o ein ne wo k has become mo e elas ic. Fig. 12 S o age modulus G0(s¼0.5 Pa) o emulsions p epa ed wi h di e en mixing aids. Final concen a ions: 1 w % HPB and F¼0.65 dodecane. Table 1 Compa ison o he d ople size (mm) o emulsions p epa ed a di e en mixing a es. Emulsion concen a ions: 1% HPB and F¼0.65 dodecane Vo ex 1000 pm 5000 pm 9000 pm D ople size/mm6034 41 18 17 894 Fig. 13 G0(Pa) a s¼0.5 Pa and ¼1 Hz measu ed a di e en ime poin s. Sample composi ion: 1% HPB and F¼0.65 dodecane, p epa ed wi h he Homo Dispe a 9000 pm. This jou nal is ªThe Royal Socie y o Chemis y 2011 So Ma e , 2011, 7, 8248–8257 | 8255 52 E idence o ilm o ma ion in he adso bed monolaye The desc ibed expe imen s ha e indica ed ha bio echnically H S a P o einsin he adso bed monolaye in he emulsions migh o m hin ilms, which means ha he indi idual molecules c osslink i e e sibly wi h each o he . The su ace ension measu emen s showed signals o i e e sible adso p ion, he C yo-TEM mic og aphs showed pieces o hin ilms, he elec ic bi e ingence measu emen s could be explained by he g ow h o la ge agg ega es and inally he la ge s o age moduli o he emulsions we e indica ions ha a s ong h ee dimensional ne wo k was o med in he emulsions. In o de o demons a e he o med h ee dimensional ne wo k, we designed an expe imen o p o e he exis ence o his ne wo k. An emulsion was p epa ed con aining 1% HPB and a mass ac ion Fo 0.65 dodecane, p epa ed wi h he Homo Dispe a 9000 pm. The emulsion was d ied in a cabine d ye a 60 C o wo weeks. La ge pieces o a li le ligh ma e ial we e ob ained. A REM-mic og aph (Fig. 14) o he ma e ial showed ha he emulsion d ople size was iden ical o he one obse ed wi h he ligh mic oscopy (Table 1). Ob iously he s uc u e had no collapsed du ing he emo al o he oil and wa e . This seems o ha e been only possible i he indi idual ilms we e c oss- linked o a supe molecula s uc u e. Emulsions om silicon oil and hyd ophobin Gel-like emulsions can no only be p epa ed om dodecane bu also om o he oils. Emulsion laye s wi h a high in e nal con en o polydime hylsiloxane (PDMS) and 0.5% HPB ha e also been p epa ed. One sample was p epa ed wi h he o ex shake while he o he samples we e p epa ed wi h a high-p essu e emulsi ie a p essu es o 100 ba , 300 ba and 1000 ba . The o ex sample and he sample p epa ed a 1000 ba sepa a ed in o wo phases: an uppe emulsion and a lowe aqueous phase. I is su p ising ha he sample which had been p oduced wi h he highes p essu e is no s able. Such si ua ions ha e also been desc ibed in he li e a u e. 22 I is usually assumed ha he e is no enough emulsi ie in he sample ha co e s he d ople s comple ely wi h a monolaye . This would also be he si ua ion in he shown sample. The dimension o he d ople dec eased as he p essu e was inc eased as is shown in Table 2. Wi h 1000 ba , a d ople diame e o abou 3 mm is eached. Wi h he simple heo e ical co e shell model (eqn (2)), one ob ains a diame e o 1 mm when a hickness o he p o ein laye o 3 nm is assumed. The iscoelas ic p ope ies o he sample inc ease wi h inc easing p essu e in he emulsi ie . I is in e es ing o no e ha he s o age modulus G0o samples wi h he same composi ion can be changed om 1 Pascal o mo e han 100 Pascal. When he concen a ion o hyd ophobin is doubled in he sample, he emulsions a e also s able a he highes p essu e used o emulsi ica ion. This expe imen shows ha he in e p e a ion o he wo phase o ma ion is p obably co ec . Mo e anspa en and single phase emulsions a e ob ained when pa o he wa e is eplaced by glyce ol as is shown in Fig. 15. These samples we e p epa ed wi h he o ex shake . The HPB concen a ion was a ied in he samples. The esul s show ha homogeneous, gel-like emulsions can al eady be ob ained wi h a p o ein concen a ion as low as 0.02%. Conclusions The in es iga ions on he p esen ed sys ems ha e shown ha emulsions om hyd ophilic su ac an s a e low iscous solu ions wi hou a yield s ess. The H S a P o eins, in con as , o m emulsions wi h gel-like p ope ies wi h a yield s ess. The gel-like p ope ies a e o med because he p o ein co e ed oil d ople s a e s icky pa icles. The s ickiness o he pa icles is due o he ac ha he amphiphilic p ope ies o he p o ein pa icles a e dis ibu ed o e hei whole su aces. This p ope y con ols also he solubili y o he p o eins in wa e . The amphiphilic p ope ies do no disappea when p o eins bind o oil d ople s. On binding he p o eins o an oil d ople , he local en i onmen on pa o he molecule is changed. As a consequence he p o ein molecule has o change i s olded s uc u e. I is concei able ha as a esul o he change o he con o ma ion, neighbou ing p o ein mole- cules in e pene a e wi h each o he and o m a hin p o ein ilm a ound he oil d ople s. This p ocess could be he eason o he aging o he emulsion and he inc ease o he shea modulus o he emulsion wi h ime. Fig. 14 REM mic og aph o he d ying esidue o an emulsion con- aining 1% HPB and F¼0.65 dodecane, p epa ed wi h he Homo Dispe a 9000 pm. Table 2 D ople size (mm) o emulsions p epa ed wi h a o ex shake and a high p essu e emulsi ie a di e en p essu es. Final concen a- ions: 0.5% HPB and F¼0.65 PDMS Vo ex 100 ba 300 ba 1000 ba D ople size/mm 100 61 4.2 0.7 3.9 1.0 3.1 0.9 Fig. 15 De e mina ion o he maximum oil con en o homogeneous emulsions depending on he used p o ein concen a ion. Aqueous phase con ained 60% glyce ol. 8256 | So Ma e , 2011, 7, 8248–8257 This jou nal is ªThe Royal Socie y o Chemis y 2011 53 Unde high shea condi ions emulsions a e ob ained in which nea ly all he p o ein is adso bed a he in e ace o he d ople s. The dimensions o he d ople s a e hen gi en by he oil/p o ein a io. The size o he d ople s in he emulsion is de e mined by he exis ing shea a es in he emulsi ie as long as enough p o ein is a ailable o co e he en i e o med oil/wa e in e ace. While no mal emulsions can be heo e ically ea ed as a dispe sion o epulsi e d ople s as i is he case o inging gels o cubic phases o which sys ems he heological p ope ies a e due o he numbe densi y o he pa icles and hei in e acial ension he emulsions om p o eins ha e o be looked a di e en ly. The p ope ies indica e ha he s o age modulus o he p o ein emulsions is de e mined by he elas ic h ee dimensional ne wo k ha su ounds he d ople s and connec s he d ople s. O he wise he high s o age moduli o he emulsions could no be unde - s ood. The elas ic ilm a ound he d ople s is p obably he eason o he high s abili y o he emulsions. The p o ein co e ed d ople s a e p esen in a loccula ed s a e wi h di ec con ac be ween he d ople s. In spi e o his si ua ion, he d ople s do no coalesce and o m an excess oil phase. Acknowledgemen s The au ho s hank he BASF AG and especially D Ul Baus o p o iding he H S a P o einsA and B o ou in es iga ions. Fu he mo e he au ho s acknowledge P o . D Dagni Danino, Technion Ins i u e o Technology in Hai a, Is ael, and he TEM- g oup o D Ma kus D echsle , Uni e si y o Bay eu h, Ge - many, o p oducing C yo-TEM mic og aphs. The au ho s mo eo e hank Ch is ian He mann om he F aunho e - Ins i u € u Silica o schung, Bay eu h, Ge many, o he p ep- a a ion o CT mic og aphs o an emulsion. Finally, special hanks o Ma ina Heide , Uni e si y o Bay eu h/BIMF, Ge - many, o g ea adminis a ion in ob aining SEM mic og aphs unde e y di icul condi ions. No es and e e ences 1 J. Johns on, Biochem. J., 1927, 21, 1314–1328. 2 M. Nino and J. M. Pa ino, J. Am. Oil Chem. Soc., 1998, 75, 1241–1248. 3 S. McClellan and E. F anses, Colloids Su ., B, 2003, 28, 63–75. 4 Y. L. Jeyachand an, E. Mielcza ski and J. A. Mielcza ski, Langmui , 2009, 25, 11614–11620. 5 S. Oh aki, H. Maeda and K. Abe, Appl. En i on. Mic obiol., 2006, 72, 2407–2413. 6 D. Yu and R. Gosh, Langmui , 2010, 26, 924–929. 7 A. Onishi and M. P oudio e, J. Sci. Food Ag ic., 1994, 65, 233–240. 8 Z. Shok ibousjein, S. Decke s and G. De delincks, Ce e isia, 2011, 35, 85–101. 9 I. Po naya, U. Cogan and D. Danino, J. Ag ic. Food Chem., 2006, 54, 5555–5561. 10 E. Me walli, J.-F. Moulin and P. M€ ulle -Buschbaum, Langmui , 2009, 25, 4124–4131. 11 D. Mye s, Su ac an Science and Technology, Wiley and Sons, 3 d edn, 2006. 12 K. A. 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This jou nal is ªThe Royal Socie y o Chemis y 2011 So Ma e , 2011, 7, 8248–8257 | 8257 54 55 Publica ion B Ma in Rege , Tomoko Sekine, Toh u Okamo o, Kei Wa anabe and Heinz Ho mann, Picke ing Emulsions s abilized by no el clay-hyd ophobin syne gism, So Ma e , 2011, 7, 11021-11030. Picke ing emulsions s abilized by no el clay–hyd ophobin syne gism Ma in Rege ,* a Tomoko Sekine, b Toh u Okamo o, b Kei Wa anabe b and Heinz Ho mann a Recei ed 8 h Augus 2011, Accep ed 15 h Sep embe 2011 DOI: 10.1039/c1sm06525d We ha e s udied he physico-chemical p ope ies o high in e nal oil in wa e (o/w) emulsions, s abilized by syne gis ic in e ac ion be ween hyd ophobin and clay. As an emulsi ying agen wi h biological backg ound we used H S a P o einB (HPB). I s emulsi ying pa ne , Laponi e XLG, is a syn he ic laye ed silica e. One o one aqueous mix u es o HPB and Laponi e XLG esul ed in homogeneous emulsions wi h an oil mass ac ion Fo 0.65 PDMS. When used sepa a ely, bo h sys ems o m uns able o/w emulsions. Mo eo e heological measu emen s indica e he weak gel-like p ope ies o hei emulsions, whe eas he simul aneous use o clay and hyd ophobin esul s in long- e m s able o/w emulsions wi h e y p onounced gel-like p ope ies. Cha ac e is ic heological p ope ies a e hei high s o age modulus G0(>1000 Pa), a high yield s ess alue and iscosi y (1 Pa s a a shea a e g¼100 s 1 ). Despi e a low polydispe si y, a ce ain ipening o he emulsion ma ix depending on he incuba ion ime and shea a e was obse ed. I is concluded ha he high s o age moduli in he gel-like emulsions a e due o he elas ici y o he clay–p o ein ilms su ounding he oil d ople s o ming a sel -suppo ing h ee-dimensional ne wo k. Ou esul s highligh he ele ance o he no el hyd ophobin–clay syne gism, esul ing in excellen ly s abilized su ac an - ee emulsions. In oduc ion P o eins a e amphiphilic compounds ha bind bo h on hyd o- philic and on hyd ophobic su aces. 1 Like o he su ace-ac i e polyme s hey also bind on clays which ha e hyd ophilic su aces. 2,3 Clays, which a e nega i ely cha ged, can be made hyd ophobic by adding ca ionic su ac an s. 4 I is usually assumed ha he binding o ca ionic su ac an s is a consequence o hei ca ionic na u e. I is known howe e ha bo h ypical non-ionic and zwi e ionic su ac an s bind o clay su aces. 5,6 By he adso p ion o hese su ac an s he su ace o clays can be u ned om hyd ophilic o hyd ophobic and back o hyd ophilic again. When he su ace o he clays becomes hyd ophobic, he clay–su ac an complexes usually p ecipi a e om aqueous solu ions. Syn he ic and na u al clays a e colloidal building blocks wi h a well-de ined s uc u e. 7 They a e laye ed silica es wi h special ion-subs i u ions, like Si by Al, Al by Mg and Mg by Li. 8 The e o e he clays possess an excess nega i e cha ge. Clay pa icles ha e a hickness o 1 nm and hei building blocks a e sepa a ed by hin laye s o ca ionic ions, usually alkali-me al ions. The clays can be ex olia ed o single shee s by applying high shea a es. Solu ions o ex olia ed clays a e anspa en and ha e a low iscosi y. Wi h inc easing concen a ion he solu ions show an ab up sol–gel ansi ion. 9 In he olde li e - a u e his ansi ion was usually explained on he basis o a ca d-house s uc u e o he gels. 10–12 I was assumed ha he nega i ely cha ged su aces o he clay shee s o m a h ee- dimensional ne wo k wi h he posi i ely cha ged sides o he clays. O he heo ies ha e been p oposed o he sol–gel an- si ions. 13 I is also concei able ha he sol–gel ansi ion is due o he in e ac ion o small s acks o clays which a e o ien ed pa allel o each o he and which become la ge wi h ime. Ve y o en he heo ies do no ake in o accoun ha he gels de elop bi e ingen p ope ies which become s onge wi h ime. Clays a e ideal compounds o he adso p ion and emo al o all kinds o was e p oduc s like dyes, mul i alen ca ions o su ac an s because hey ha e such huge su aces o up o 1000 m 2 g 1 . 14,15 Clay–su ac an complexes a e pe ec sys ems o he p epa- a ion o Picke ing emulsions. Such emulsions om clay/non- ionic su ac an sys ems ha e p o en o be qui e s able. 16 In his a icle we p epa e Picke ing emulsions om clay–p o ein complexes and compa e he p ope ies o hese emulsions o emulsions which a e p epa ed om he p o eins alone. The emulsions we e o med wi h a ecombinan ly p oduced hyd o- phobin, called H S a P o ein. 17 I is p oduced as usion p o ein ha bo ing he hyd ophobin p o ein o he ungi Aspe gillus nidulans. Hyd ophobins ac as highly su ace-ac i e p o eins 18,19 and a e well known o hei s ong endency o sel - agg ega e. 20,21 These p ope ies combined wi h i s now ob ained high a ailabili y due o gene ic enginee ing make he H S a P o einin e es ing o indus ial applica ions. a Uni e si y o Bay eu h, BZKG/BayColl, Go lieb-Keim-S aße 60, 95448 Bay eu h, Ge many. E-mail: [email p o ec ed] b Shiseido Resea ch Cen e , 2-2-1 Hayabuchi, Tsuzuki-ku, Yokohama, 224- 8558, Japan. E-mail: [email p o ec ed] This jou nal is ªThe Royal Socie y o Chemis y 2011 So Ma e , 2011, 7, 11021–11030 | 11021 Dynamic A icle LinksC < So Ma e Ci e his: So Ma e , 2011, 7, 11021 www. sc.o g/so ma e PAPER 56 Ma e ials and me hods H S a P o einB, om now abb e ia ed as HPB (19 kDa; IEP: 6.15), is a ecombinan hyd ophobin 17 and was a gi om BASF, Ludwigsha en. HPB consis s o he class I hyd ophobin DewA om he ungi Aspe gillus nidulans and he Bacillus sub ilis p o ein yaaD, espec i ely, a unca ed o m o yaaD. Fo mo e de ailed in o ma ion abou he H S a P o einB please e e o e . 17. The clay Laponi e XLG 22 was pu chased om Rockwood Clay Addi i es GmbH, Moosbu g. Polydime hylsiloxane (PDMS) was pu chased om Shine su Kagaku, Tokyo. I has gene al o mu- la ion: (CH 3 ) 3 SiO[(CH 3 )2SiO]nSi(CH 3 ) 3 . The polyme iza ion deg ee his anging om 5 o 19 (>98%) and he iscosi y is app oxima ely 6 mPa s. Me ck, Da ms ad , supplied he nonpola oils decane and dodecane, as well as he pola oil oc yl-me hox- ycinnama e (OMC, b and name: Eusolex2292). O he chem- icals no speci ied in he ex we e o analy ical g ade o equi alen . The su ace ension so he samples was measu ed wi h he olume-d op ensiome e TVT1 om Lauda Co., K€ onigsho en, a a cons an d op- o ma ion speed o 1 mls 1 . In o de o de e mine he ee amoun o hyd ophobin in he clay–hyd o- phobin mix u es he supe na an o he samples was used. The e o e he samples we e cen i uged in a Medi uge om He aeus Ins umen s GmbH, Hanau, o 10 minu es a 2000g. Fo C yo-T ansmission Elec on Mic oscopy (C yo-TEM) a d op o he sample was placed on a TEM-g id (200 mesh, Science Se ices, Munich). Remo ing he majo i y o he liquid sample wi h blo ing pape esul ed in a hin s e ched ilm o e he g id holes. A e wa ds he specimens we e shock- i i ied by apid imme sion in o liquid e hane and cooled o below 178 C by liquid ni ogen in a Zeiss C yobox eezing uni . The speci- mens, kep below 178 C, we e s udied in a Zeiss EM922 Omega EFTEM ansmission elec on mic oscope, ope a ed a 200 kV. All images we e digi alized wi h he CCD came a sys em om Ul ascan 1000, Ga an. All emulsions we e p epa ed om aqueous solu ions o hyd ophobin and clay. In o de o a oid mic obial g ow h all samples con ained 0.5 w % phenoxye hanol. I u ned ou ha one s ep oil addi ion o he aqueous phase led o emulsion b eakdown, so i was only possible o p oduce high oil con en emulsions wi h s epwise addi ion o oil. Samples emulsi ied wi h a Vo ex shake (IKA Genius 3, S au en) we e ea ed o 0.5 h a maximum powe . High p essu e emulsions we e p e-emulsi ied using he Homo Dispe (Tokushu Kika, Osaka) wi h abou 1000 e olu ions pe minu e ( pm). A e wa ds he p e-emulsions we e illed in he High P essu e Emulsi ie (APV 1000, Albe slund) and passed h ee imes h ough he de ice a he desi ed p essu e (100–1000 ba ). Fo ligh mic oscopy he samples we e apped be ween a mic oscope slide and co e glass and in es iga ed wi h a Zeiss ligh mic oscope (model: 47 60 05-9901). The mic og aphs we e digi alized wi h he DFK 41F02 came a and analyzed wi h he IC cap u e 2.1 so wa e (The Imaging Sou ce, B emen). The heology o he emulsion laye s was measu ed wi h a cone- pla e heome e RheoS ess 600 om Haake The mo Scien i ic, Ka ls uhe a 25 C. The expe imen al da a we e analyzed wi h he Haake RheoWin Da a Manage , Ve sion 3.3. Fo C yo-Scanning Elec on Mic oscopy (C yo-SEM) he sample was apped in aluminium specimens and apidly ozen in liquid ni ogen in he Leica BalTec HFM-100 eeze de ice. Using he Leica EM VCT 100 Vacuum-C yo-T ans e -Sys em he sample was loaded unde cold ni ogen a mosphe e in he Leica EM MED 020 eeze ac u e and spu e de ice. A e cu ing he specimens by a ca bide me al kni e, hey we e immedia ely co e ed wi h a pla inum laye o desi ed hickness. Finally he Ul a Plus Zeiss SEM ha bo ing a hi d-gene a ion Gemini elec on op ical column was cha ged wi h he coa ed specimens. The in eg a ed The mo Scien i ic MagnaRay WDS spec ome e au oma ically handled alignmen , analysis se ings and da a acquisi ion and eased he measu emen p ocedu e. The emulsion was illed in a ound-bo om lask and a ached o he F eeze D ye ALPHA 1-4 om Ch is GmbH, Os e ode, un il all o he wa e and oil had been emo ed. The eeze-d ied emulsion was coa ed wi h a 1.3 nm i on laye in he C essing on Spu e Coa e 208 HR and analyzed wi h a Zeiss 1530 scanning elec on mic oscope (SEM). Resul s and discussion In e ac ion o clay and hyd ophobin Solu ions o clays and o he hyd ophobin a e low iscous and anspa en . Mix u es o he wo compounds a e u bid howe e (Fig. 1). The u bidi y could be due o deple ion loccula ion because bo h pa icles ca y he same nega i e ionic cha ge bu a e o di e en size and shape. On he basis o su ace ension measu emen s deple ion loccula ion can be uled ou because he ee p o ein concen a ion is e y much lowe han he o al p o ein concen a ion in he mixed samples. The u bidi y mus he e o e be due o agg ega es be ween he wo pa icles e en hough he long ange in e ac ion be ween he pa icles is epulsi e. The samples o he mix u es look e y simila o mix u es be ween clays and poly inyl alcohol (PVA). In such samples i had been shown ha PVA adso bed on o clay pa icles and he o med complexes agg ega e in solu ions o la ge clus- e s. 23 Ob iously he si ua ion be ween Laponi e XLG and HPB is simila o he si ua ion be ween clays and PVA. In bo h cases he polyme s bind o he clay su ace. The adso p ion ene gy o he binding o he nega i ely cha ged hyd ophobin o he nega- i ely cha ged clay pa icles can o e come he epulsi e in e ac- ion ene gy be ween he wo pa icles. I is in e es ing o no e ha he samples wi h mixing a ios o 8 : 2 and 3 : 7, 2 : 8 and 1 : 9 ha e sepa a ed in o wo laye sys ems while he o he samples a e u bid bu ha e no sepa- a ed on a mac oscopic le el. The dependence o he amoun o hyd ophobin adso bed o 0.5 w % Laponi e XLG on he used hyd ophobin concen a ion was de e mined as ollows. HPB solu ions in he p esence and absence o 0.5 w % clay we e p epa ed. The samples con aining hyd ophobin and clay we e cen i uged. As clay pa icles co e ed wi h hyd ophobin o med a p ecipi a e, he emaining, non- adso bed HPB was loca ed in he supe na an . By de e mining he su ace ension o he supe na an and compa ing i o he alues o HPB wi hou clay, he amoun o non-adso bed HPB could easily be ob ained. Consequen ly he o he pa o he ini ial HPB amoun was adso bed on o he clay pa icles. Su ace ension measu emen s a e shown in Fig. 2. 11022 | So Ma e , 2011, 7, 11021–11030 This jou nal is ªThe Royal Socie y o Chemis y 2011 57 dimensions o he d ople s in he emulsions. Li le a en ion was usually paid o he heological p ope ies o he emulsions. I is he e o e di icul o compa e he p ope ies o he p e iously ob ained esul s wi h he p ope ies o his in es iga ion. Picke ing emulsions om clays and su ac an s ha e been p epa ed by he g oup o Lagaly. 16,42 I was shown ha non-ionic su ac an s like glyce ol monos ea a e C 16 E 10 , suga su ac an and leci hin bind o clays and s able o/w emulsions wi h mo e han 50% o oil could be ob ained. I was men ioned ha he emulsions showed hixo opic beha io which means ha he emulsions had a yield s ess. The s o age modulus o he emul- sions was no measu ed and no in o ma ion was gi en ega ding he eason o he hixo opic beha iou . No linea iscoelas ic egion was obse ed. While no de ailed compa ison can be made he gene al ema ks in he in es iga ion indica e ha he p op- e ies o he emulsions did no ha e such a s ong gel-like beha iou as obse ed in his in es iga ion. De ailed s udies on Picke ing emulsions om umed silica pa icles and ca ionic su ac an s we e epo ed om he g oup o B. P. Binks. 43 In con as o he clay pa icles which ha e a su ace a ea a ound 1000 m 2 g 1 , he used silica pa icles ha e only a su ace o a ound 250 m 2 g 1 . In he dispe sed s a e he pa icles a e nega i ely cha ged. Thei isoelec ic poin is below pH 3. The pa icles could be dispe sed in wa e o clea o bluish solu ions. Wi h inc easing ca ionic su ac an p ecipi a ion o he silica– su ac an complexes occu ed. The complexes did no e- dissol e wi h excess su ac an . Emulsions could be p epa ed om hese complexes wi h a wa e /oil a io o 1 : 1. I was men ioned ha he mos s able emulsions we e ob ained a he condi ions whe e he amoun o p ecipi a e had a maximum. SEM measu emen s on he emulsions showed ha he silica– su ac an complexes we e no e enly dis ibu ed on he su ace o he d ople s and he d ople s we e conside ably la ge as in his in es iga ion. A highe silica concen a ion had o be used han clays in his in es iga ion o each s able emulsions. In iew o he la ge pa icles his is no su p ising. No heo- logical measu emen s we e made on he emulsions. The SEM mic og aphs o he dilu ed emulsions indica ed howe e ha he in e ac ion be ween he d ople s was a ac i e. Picke ing emulsions ha e also been p epa ed om needle-like pa icles as om su ac an coa ed Boehmi e. 37 These pa icles a e posi i ely cha ged and ha e a hickness o a ound 20 nm. In spi e o hei la ge dimensions s able emulsions could be ob ained wi h as li le as 0.05% o Boehmi e. The d ople s had a diame e o 20 mm o mo e. No heological p ope ies o hese emulsions we e epo ed. These commen s on a ailable in es iga ions on Picke ing emul- sions make i likely ha he s udied emulsions did no ha e such gel-like p ope ies as he emulsions o his in es iga ion whe e he gel-like p ope ies a e mos p ominen and in con as o no mal emulsions which e y o en ha e a low iscous beha iou . As a as we know ou in es iga ion is he i s one in which i was men ioned ha he s abili y o he emulsion is due o a sel -suppo ing h ee- dimensional ne wo k om he clay/p o ein pa icles. Conclusions Hyd ophobins, in bo h he nega i ely cha ged and in he posi- i ely cha ged s a e, bind s ongly o clay pa icles which a e dispe sed in aqueous solu ions. The esul ing clay–hyd ophobin compounds can be used as emulsi ie s o he o ma ion o homogeneous o/w emulsions wi h oil con en be ween 30 and 65% by weigh . The emulsions a e s abilized in a wo s age p ocess. Fi s ly he p o ein–clay pa icles a e highly su ace ac i e due o he p ope ies o hyd ophobin and he e o e p e en he eshly o med oil d ople s om coalescence. In he nex s ep he hyd ophobin–clay ne wo k e ol es due o hyd o- phobin–hyd ophobin en anglemen and in e ac ion wi hin one day and he e o e p o ides long e m s abili y o he p oduced emulsions. The emulsions ha e gel-like p ope ies because he hyd ophobin-sandwiched clay pa icles ac as s icky pa icles and o m h ee-dimensional ne wo ks in he emulsions. The elas ic p ope ies o he gels a e due o he h ee-dimensional ne wo k o he clay–hyd ophobin ne wo k. The oil and wa e can be emo ed om he emulsions by eeze-d ying wi hou collapse o he ne wo k s uc u e. Acknowledgemen s The au ho s g a e ully hank he BASF SE and especially D U. Baus o p o iding he H S a P o einB. Mo eo e he au ho s hank he TEM-g oup o D M. D echsle , Uni e si y o Bay - eu h, Ge many, o p oducing C yo-TEM mic og aphs o he samples. Finally special hanks o D B. F€ o s e and M. Heide , Uni e si y o Bay eu h/BIMF, Ge many, o immense adminis- a ion a ob aining C yo-SEM and SEM mic og aphs unde challenging condi ions. No es and e e ences 1 Y. Jeyachand an, E. Mielcza ski and J. Mielcza ski, Langmui , 2009, 25, 11614–11620. 2 K. Ralla, U. Sohling and T. Schepe , Biop ocess Biosys . Eng., 2010, 33, 847–861. 3 J.-M. 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Yo k, Langmui , 2011, 27, 2357–2363. 41 B. Tigges, T. Dede ichs and O. Weichold, Langmui , 2010, 26, 17913– 17918. 42 G. Lagaly, M. Reese and S. Abend, Appl. Clay Sci., 1999, 14, 279– 298. 43 B. P. Binks, J. A. Rod igues and W. Fi h, Langmui , 2007, 23, 3626– 3636. 11030 | So Ma e , 2011, 7, 11021–11030 This jou nal is ªThe Royal Socie y o Chemis y 2011 65 66 Publica ion C Ma in Rege and Heinz Ho mann, Hyd ophobin coa ed Boehmi e Nanopa icles s abilizing oil in wa e emulsions, J. Colloid In e ace Sci., 2011, doi: 10.1016/j.jcis.2011.10.050. Hyd ophobin coa ed boehmi e nanopa icles s abilizing oil in wa e emulsions Ma in Rege ⇑ , Heinz Ho mann Uni e si y o Bay eu h, BZKG/BayColl, Go lieb-Keim-S aße 60, 95448 Bay eu h, Ge many a icle in o A icle his o y: Recei ed 31 Augus 2011 Accep ed 18 Oc obe 2011 A ailable online xxxx Keywo ds: Boehmi e Hyd ophobin Nanopa icles Picke ing emulsion Syne gism abs ac Hyd ophobin coa ed boehmi e nanopa icles ha e been used o es ablish oo h-pas e like, homogenous emulsions. The su ace-modified nanopa icles we e simply ob ained by mixing aqueous solu ions o ca - ionic boehmi e pa icles wi h he anionic hyd ophobin H S a P o ein B Ò (HPB). Su ace ension measu e- men s clea ly show ha 1 w .% boehmi e binds up o 1 w .% HPB. The s ong in e ac ion and agg ega ion o hyd ophobin coa ed boehmi e nanopa icles was p o en by C yo-TEM measu emen s, oo. In e es ingly, he combined use o 0.5 w .% HPB and 0.5 w .% boehmi e as emulsi ying agen s esul ed in e y s able, homogenous, high in e nal phase emulsions (65 w .% oil) ha a e s able o e mon hs. The es ablished emulsions ha e also been cha ac e ized by heological measu emen s. S o age moduli o mo e han 1000 Pa a e cha ac e is ic o hei high gel-like p ope ies. Fu he mo e, ligh mic oscopy showed an a e - age d ople size close o 1 l m wi h low polydispe si y. C yo-SEM confi med ha he hyd ophobin coa ed nanopa icles a e loca ed a he in e ace o he oil d ople s and he e o e s abilize he emulsion sys ems. Ó2011 Else ie Inc. All igh s ese ed. 1. In oduc ion The e is cu en ly g ea scien ific in e es a ound hyd ophobins and hei possible applica ions [1–4]. Hyd ophobins a e iny, cys eine- ich p o eins ha a e buil up om only abou 100 amino acids. They a e ac ing as highly su ace ac i e p o eins [5,6] and a e well known o hei s ong endency o sel -agg ega ion [7,8] e en a in e aces [9]. They a e now a ailable in kilog am scale due o gene ic enginee ing [10]. In na u e, filamen ous ungi p oduce hyd ophobins o se e al easons, like o he ungal a achmen o hyd ophobic su aces [11]. Based on al e ed p ope - ies ega ding hei s uc u e and unc ion, hyd ophobins ha e been classified in ype I and II [12]. In his s udy we use H S a P o- ein B Ò (HPB) [10]. I is based on class I hyd ophobin DewA om Aspe gillus nidulans. DewA is connec ed o a usion pa ne , espec i ely a unca ed o m o yaaD om Bacillus sub ilis. Recen ly i was shown ha o/w emulsions p epa ed wi h HPB as emulsifie ha e gel-like p ope ies e en a as low concen a ions as 0.2 w .%. The emulsions ha e long- e m s abili y [13]. I was concluded ha he hyd ophobin coa ed oil d ople s ac as s icky d ople s and a e a ac ing each o he . The hyd ophobin molecules adso bed a di e en oil d ople s in e ac , pene a e and en angle wi h each o he . As a consequence, a h ee dimensional p o ein ne wo k h ough he whole emulsion sample is o med. A scanning elec on mic oscope mic og aph o he d ying esidue o an emulsion con aining 1% HPB and 65% dodecane confi med his assump ion [13]. The g oup o Lips ecen ly obse ed simila e ec s o class II hyd ophobin in aqueous solu ion. They epo ha a sig- nifican binding ene gy be ween wo hyd ophobin molecules ex- is s. This a ac ion is due o sho - ange hyd ophobic in e ac ion [14,15]. In he case o p o eins as emulsi ying agen s, like ß-lac oglobulin, i is usually assumed ha he p o ein molecules o m beside a s e ical ba ie also a epulsi e one be ween he oil d ople s [16–18]. Ob iously emulsions based on hyd ophobin a e s abilized by a ac i e in e ac ion be ween he s icky emulsion d ople s. Clay mine als a e in e es ing compounds o di e en applica- ions [19,20] and ha e ecen ly been used in combina ion wi h HPB as emulsifie [21]. In his combina ion, e en hough bo h pa icles a e nega i ely cha ged, su ace ension measu emen s clea ly indica ed a s ong binding be ween hem. One o one aque- ous mix u es o HPB and Laponi e XLG in combina ion wi h all ypes o oils esul ed in e y s able, homogenous o/w emulsions wi h ex ao dina ily gel-like p ope ies. A high s o age modulus G 0 and iscosi y a e cha ac e is ic heological p ope ies o hese emulsions. I was concluded ha he elas ici y o he sample is due o he o ma ion o a sel -suppo ing h ee dimensional ne - wo k by he p o ein–clay pa icles. A e emo ing he oil and wa e by eeze d ying he p o ein–clay ne wo k emained. La ely Tigges e al. in es iga ed he p ope ies o emulsions s abilized wi h su ace-modified boehmi e pa icles [22]. Boehmi e is an alumina powde wi h a ca ionic su ace. In ha s udy, i was modified wi h p-dodecylbenzenesul onic acid as ionic and p- oluensul onic acid as non-ionic su ac an . Picke ing emulsions p epa ed om hese combina ions we e no s able o longe han 0021-9797/$ - see on ma e Ó2011 Else ie Inc. All igh s ese ed. doi:10.1016/j.jcis.2011.10.050 ⇑ Co esponding au ho . E-mail add ess: [email p o ec ed] (M. Rege ). Jou nal o Colloid and In e ace Science xxx (2011) xxx–xxx Con en s lis s a ailable a SciVe se ScienceDi ec Jou nal o Colloid and In e ace Science www.else ie .com/loca e/jcis Please ci e his a icle in p ess as: M. Rege , H. Ho mann, J. Colloid In e ace Sci. (2011), doi:10.1016/j.jcis.2011.10.050 67 a ew days and showed ypical emulsion ins abili y signs like c eaming. In his pape , howe e , i will be shown ha e y s able, homogenous and oo h-pas e like Picke ing emulsions, ha o e long e m s abili y, can be p epa ed by he use o small amoun s o boehmi e pa icles in combina ion wi h hyd ophobin. Rheologi- cal measu emen s o he ob ained Picke ing emulsions indica e hei high gel-like cha ac e . The low amoun o emulsifie as well as he ou s anding s abili y makes hese emulsions no only e y in e es ing o science bu also o indus ial applica ion, like in cosme ics. The sec ion wi h he esul s o his manusc ip is o ganized as ollows. The fi s pa is abou he cha ac e iza ion o boehmi e ega ding issues ha a e impo an o a deepe unde s a emen o ou u he in es iga ions. The second sec ion pa deals wi h he hyd ophobin–boehmi e in e ac ion. The amoun o adso bed hyd ophobin in dependency o he used concen a ion will be mon- i o ed by su ace ension measu emen s. Mo eo e C yo-TEM s ud- ies o boehmi e wi hou and wi h hyd ophobin a e shown, p o ing clea ly hei in e ac ion. The hi d pa is abou Picke ing emulsions p epa ed om hyd ophobin coa ed boehmi e pa icles. We de e - mined he heological p ope ies o hese emulsions. A ce ain ip- ening o he p o ein–boehmi e ma ix in dependency o s o age ime will be epo ed. The long- e m s abili y o he emulsions will be confi med by obse ing he emulsion homogenei y as well as he emulsion d ople size in dependency o s o age ime. Finally, he ou h pa shows he e ec s o a ying di e en pa ame e s, like he p o ein concen a ion o oil pola i y, o he appea ance and he- ological cha ac e is ics o he Picke ing emulsions. 2. Ma e ials and me hods 2.1. Ma e ials H S a P o ein Ò B, om now abb e ia ed as HPB (19 kDa; IEP: 6.15; Ze a Po en ial – 31 mV o 1 w .% solu ion), is a ecombinan hyd ophobin [10] and was a gi om BASF, Ludwigsha en. The com- me cial boehmi e powde , called Dispe al P2, was pu chased om Sasol GmbH, Hambu g. Boehmi e is a c –AlO(OH) wi h a ca ionic su ace. Se a Elec opho esis GmbH, Heidelbe g, p o ided he an- ionic su ac an Sodium-Dodecylsul a e (SDS). Polydime hylsilox- ane (PDMS) was pu chased om Shine su Kagaku, Tokyo. I has gene al o mula ion: (CH 3 ) 3 SiO[(CH 3 )2SiO]nSi(CH 3 ) 3 . The polyme - iza ion deg ee g is anging om 5 o 19 (>98%) and he iscosi y is app oxima ely 6 mPa s. Me ck, Da ms ad , supplied he nonpola oil Dodecane, as well as he pola oil Oc yl-me hoxycinnama e (OMC, b and name: Eusolex Ò 2292). O he chemicals no specified in he ex we e o analy ical g ade o equi alen . 2.2. Su ace ension The su ace ension o he samples was measu ed wi h he olume-d op ensiome e TVT1 om Lauda Co., Königsho en, a a cons an d op- o ma ion speed o 3 s/ l l. In o de o de e mine he ee amoun o p o ein in he boehmi e–hyd ophobin mix u es, he supe na an o he samples was used. The samples we e he e- o e cen i uged in a Medi uge om He aeus Ins umen s GmbH, Hanau, o 10 min a 2000g. 2.3. C yo-TEM Fo C yo-T ansmission Elec on Mic oscopy (C yo-TEM) a d op o he sample was placed on a TEM-g id (200 mesh, Science Se ices, Munich). Remo ing he majo i y o he liquid sample wi h blo ing pape esul ed in a hin s e ched film o e he g id holes. A e wa ds he specimens we e shock- i ified by apid imme sion in o liquid e hane and cooled o below 178 °C by liquid ni ogen in a Zeiss C yobox eezing uni . The specimens, kep below 178 °C, we e s udied in a Zeiss EM922 Omega EFTEM ansmission elec on mic oscope, ope a ed a 200 kV. All images we e digi alized wi h he CCD came a sys em om Ul ascan 1000, Ga an. 2.4. Emulsion p epa a ion All emulsions we e p epa ed om aqueous solu ions o hyd ophobin and boehmi e. As an imic obial agen , all samples con ained 0.5 w .% phenoxye hanole. I was only possible o p o- duce high oil con en emulsions wi h s epwise addi ion o oil. Sam- ples emulsified wi h Vo ex shake (IKA Genius 3, S au en) we e ea ed o 0.5 h a maximum powe . High p essu e emulsions we e p e-emulsified using he Homo Dispe (Tokushu Kika, Osaka) wi h abou 1000 pm ( pm). A e wa ds he p e-emulsions we e filled in he High P essu e Emulsifie (APV 1000, Albe slund) and passed h ee imes h ough he de ice a he desi ed p essu e (100– 1000 ba ). 2.5. Ligh mic oscopy The samples we e apped be ween a mic oscope slide and co e glass and in es iga ed wi h a Zeiss ligh mic oscope (model: 47 60 05-9901). The mic og aphs we e digi alized wi h he DFK 41F02 came a and analysed wi h he IC cap u e 2.1 so wa e (The Imaging Sou ce, B emen). 2.6. Rheology The heology o he emulsion laye s was measu ed wi h a cone- pla e heome e RheoS ess 600 om Haake The mo Scien ific, Ka ls uhe a 25 °C. The expe imen al da a we e analysed wi h he Haake RheoWin Da a Manage , Ve sion 3.3. 2.7. C yo-SEM Fo C yo-Scanning Elec on Mic oscopy (C yo-SEM) he sample was apped in aluminum specimens and apidly ozen in liquid ni ogen in he Leica BalTec HFM-100 eeze de ice. Using he Leica EM VCT 100 Vacuum-C yo-T ans e -Sys em he sample was loaded unde cold ni ogen a mosphe e in he Leica EM MED 020 eeze ac u e and spu e de ice. A e cu ing he specimens by a ca bide me al kni e, hey we e immedia ely co e ed wi h a pla inum laye o desi ed hickness. Finally he Ul a Plus Zeiss SEM ha bo ing a hi d gene a ion Gemini elec on op ical column was cha ged wi h he coa ed specimens. The in eg a ed The mo Scien ific MagnaRay WDS spec ome e au oma ically handled alignmen analysis. 3. Resul s and discussions 3.1. Cha ac e iza ion o boehmi e In he case o clays, i is basic scien ific knowledge o ha e in o - ma ion abou hei ca ionic exchange capaci y (cec). Fo example, he nega i ely cha ged clay Laponi e XLG has a ca ionic cec o 65.7 meq/100 g [23]. By adding di e en amoun s o ca ionic su - ac an , one can easily adjus he cha ge ype and ex en o he mix u e. Such impo an in o ma ion, howe e , was s ill missing in he case o he posi i ely cha ged boehmi e nanopa icles. The de e mina ion o he anionic exchange capaci y (aec) o 1 w .% boehmi e was done as ollows. Samples om 1 w .% boehmi e wi h inc easing concen a ions o he anionic su ac an SDS ha e been p epa ed. By moni o ing he su ace ension beha io o he 2M. Rege , H. Ho mann / Jou nal o Colloid and In e ace Science xxx (2011) xxx–xxx Please ci e his a icle in p ess as: M. Rege , H. Ho mann, J. Colloid In e ace Sci. (2011), doi:10.1016/j.jcis.2011.10.050 68 supe na an s o he boehmi e–SDS mix u es, he aec could easily be de e mined (Fig. 1). The aec o boehmi e is de e mined o 50 meq/100 g. Acco ding o Fig. 1, up o 35 mM SDS can bind o 1 w .% boehmi e. Clays a e also well known o p o iding an ab up sol–gel ansi- ion wi h inc easing concen a ion o he solu ions [24]. Conse- quen ly he iscosi y g in dependency o he clay concen a ion inc eases a he sol–gel ansi ion poin apidly. Fo ou s udies, i is impo an o check, whe he some hing simila like his can be obse ed in pu e boehmi e solu ions. We chose he e o e a concen- a ion ange o 0.5–10 w .% boehmi e. To he naked eye he solu- ions appea ed o be low iscous. In o de o ge exac alues, he iscosi y g was de e mined by oscilla ing measu emen s. Table 1 o - e s an o e iew o he iscosi y g a a equency o 1 Hz a wo di - e en shea s esses s (0.05 and 0.5 Pa) o 0.5–10 w .% boehmi e solu ions. The esul s summa ized in Table 1 show clea ly ha boehmi e solu ions in his concen a ion ange a e shea hinning. E en wi h 10 w .% boehmi e he iscosi y is only eigh imes highe han he one o wa e . Mo eo e equency dependen measu emen s o he solu ions did no show any sign o elas ic beha io e en a e y low shea s esses (<0.05 Pa). Acco dingly he boehmi e solu ions show no sol–gel ansi ion in he used concen a ion ange. 3.2. In e ac ion o boehmi e and hyd ophobin 1 w .% solu ions o boehmi e and hyd ophobin (HPB) a e ans- pa en and low iscous. Mix u es o he wo compounds howe e show bicon inuous beha io wi h a swollen, u bid lowe phase (Fig. 2). The u bidi y o he mix u es mus be due o agg ega es o med by he wo pa icles. As bo h pa icles ca y opposi e cha ges, hey ob iously in e ac wi h each o he . The hyd ophobin adso bs on he boehmi e pa icles due o elec os a ic a ac ion. The p ecipi- a es a e no compac , bu somehow swollen. We assume he hyd ophobin co e ed boehmi e uni s as s icky pa icles. Beside ou p e ious in es iga ions [13,21], o he scien is s ha e shown ha hyd ophobins sel -agg ega e and o m monolaye s [14,15]. We imagine ha he hyd ophobin does no loose hese abili ies due o adso p ion on a solid su ace. I could he e o e be possible ha he pa icles a e o ming a loose h ee-dimensional ne wo k. In o de o p o e his assump ion, oscilla ing, heological measu e- men s o he swollen p ecipi a e a e cen i uga ion (2000 pm, 5 min) o he 1:1 mix u e (Fig. 2) ha e been pe o med. I u ned ou ha he hyd ophobin coa ed boehmi e pa icles uly o m a h ee-dimensional ne wo k. Fo de ailed in o ma ion conside he Suppo ing In o ma ion pa . One o he mos swollen samples, espec i ely he 1:1 mix u e, will be aken as emulsifie o he o - ma ion o Picke ing emulsions in Sec ion 3 o he esul pa . Su ace ension measu emen s o HPB solu ions wi hou and in he p esence o 0.5 w .% boehmi e a e shown in Fig. 3. The esul s om he figu e allow de e mining he adso bed and ee amoun o hyd ophobin a di e en concen a ions. The esul s a e summa ized in Table 2. The esul s in Table 2 show ha 0.5 w .% boehmi e can bind up o 0.5 w .% hyd ophobin. The su ace ension measu emen s indi- ca e ha he samples s ill con ain some ee p o ein when he o al amoun o p o ein and o boehmi e is 0.5 w .%. Ano he impo an aspec comes up, i we emembe he anionic exchange capaci y (aec; 50 meq/100 g) o he boehmi e. A concen a ion o 2.5 mM SDS is enough o compensa e he cha ge o 0.5 w .% boehmi e. Re e ing o Fig. 3 0.1 w .% HPB bind o 0.5 w .% boehmi e be o e he su ace ension s a s o dec ease. 0.1 w .% HPB co esponds o a mola concen a ion o 0.05 mM. Conside ing i s amino acid composi ion, HPB con ains fi e aspa ic acid as well as wel e glu amine acid esidues. I we assume all o hem o be cha ged, he mola concen a ion would ise o 0.85 mM. E en his concen a ion is ob ious no close o he aec. In o de o unde s and his appa en disc epancy, one has o emind he di e ences be ween p o eins and su ac an s. The size o he olded HPB is abou 5 nm [13]. Consequen ly, he equi ed space pe molecule o adso p ion is much bigge compa ed o he su ac- an SDS. The big size has wo e ec s. On he one hand one p o ein molecule p obably masks mo e han one posi i e cha ge o he boehmi e. On he o he hand s e ical e ec s may play an impo an ole p e en ing o he p o ein molecules om adso p ion. Mo eo e he possibili y o con o ma ional ea angemen upon adso p ion o he p o ein in o de o ge a be e elec os a ic in e ac ion wi h he boehmi e is possible. In Fig. 4 C yo-TEM mic og aphs a e shown om solu ions o boehmi e and o solu ions o HPB co e ed boehmi e nanopa icles. The needle-like boehmi e pa icles ha e sizes be ween 10 and 50 nm and a e homogeneously dis ibu ed o e he whole a ea in he pu e boehmi e sample. The boehmi e pa icles in he hin film seem o show almos only pa allel o pe pendicula o ien a ion o he film. These limi ed o ien a ions a e likely o be a esul o he elec os a ic epulsion be ween he boehmi e pa icles. In he p es- ence o equal amoun s o hyd ophobin, he boehmi e pa icles a e clus e ed in o domains o 1 l m size. Mo eo e he hyd ophobin coa ed boehmi e nanopa icles seem o sha e many b idging poin s wi h each o he . These mul iple connec ions a e an indica ion o he s icky cha ac e o he modified boehmi e pa icles. Conside - ing he C yo-TEM mic og aphs, one can easily imagine he o ma ion o a sel -suppo ing h ee-dimensional ne wo k by he hyd ophobin co e ed boehmi e pa icles. 3.3. Syne gis ic emulsi ying ac ion o hyd ophobin modified boehmi e In his chap e he influence o he used emulsifie ype o he p ope ies o he ob ained emulsions will be in es iga ed and com- pa ed o each o he . All emulsions he e o e ha e been p epa ed Fig. 1. Su ace ension p ofile o he supe na an s o samples wi h 1 w .% boehmi e and inc easing amoun s o he su ac an SDS in compa ison o he su ace ension o SDS alone. The anionic exchange capaci y o 1% boehmi e is 5 mM SDS. Table 1 O e iew o he iscosi y g a wo di e en shea s esses s (0.05 and 0.5 Pa) de e mined by oscilla ing measu emen s o boehmi e solu ions in he concen a ion ange 0.5–10 w .%. [Single column]. Boehmi e (w .%) 0.5 1 2.5 5 7.5 10 g (mPa s) a s = 0.05 Pa 7.9 10.7 15.3 16.9 25.9 44.1 g (mPa s) s = 0.5 Pa 5.4 4.8 5.7 5.2 6.7 7.9 M. Rege , H. Ho mann / Jou nal o Colloid and In e ace Science xxx (2011) xxx–xxx 3 Please ci e his a icle in p ess as: M. Rege , H. Ho mann, J. Colloid In e ace Sci. (2011), doi:10.1016/j.jcis.2011.10.050 69 om wa e and silicon oil (PDMS) unde he same condi ions bu wi h di e en emulsifie s. In Fig. 5 emulsions p epa ed wi h a high p essu e emulsifie a a p essu e o 1000 ba and an oil mass ac- ion U o 0.65 PDMS a e shown. The used emulsifie s we e 0.5 w .% HPB (a), 0.5 w .% boehmi e (b) and 0.5 w .% HPB in combina ion wi h 0.5 w .% boehmi e (c). The samples we e pho og aphed 1 day a e p epa a ion. The sample wi h hyd ophobin as emulsi ying agen is a wo laye sys em wi h an uppe gel-like emulsion and a lowe aqueous laye . Fo me in es iga ions confi med hese emulsions o be o he o/w ype [13]. Rheological measu emen s showed ha he hyd ophobin based emulsions ha e gel-like p ope ies due o he o ma ion o a sel -suppo ing h ee-dimensional ne wo k. In ac , he emulsion shown in Fig. 5a is no a homogenous one. The wo laye si ua ion is p obably due o he ac ha he hyd ophobin concen a ion is no high enough o co e comple ely he su ace o he eshly shea ed, smalle oil d ople s. Using lowe p essu es (100 o 300 ba ) esul ed in homogeneous emulsions. The emulsion ob- ained om he pu e boehmi e (Fig. 5b) is an inhomogeneous, mul- ilaye emulsion. I sepa a es wi hin one day in h ee laye s, a lowe aqueous laye , an aqueous emulsion and a e y small, uppe oily laye ha can ha dly be seen in Fig. 5b. I is no iceable, ha a solu- ion o 0.5 w .% boehmi e has a su ace ension o 72.80 mN/m indi- ca ing i s e y hyd ophilic cha ac e , bu i can ac a leas as weak emulsi ying agen . Fascina ingly, he emulsion based on 0.5 w .% boehmi e co e ed wi h 0.5 w .% HPB is a homogenous, oo h-pas e like emulsion (Fig. 5c). As i is al eady ob ious om he pic u e, he oil d ople s in he emulsion mus be held in a h ee-dimensional ne wo k buil up by he hyd ophobin co e ed boehmi e pa icles. They ac as s icky pa icles ha a e in e ac ing wi h each o he gi - ing he emulsion gel-like p ope ies. In o de o compa e he heological p ope ies o he emulsion laye s om Fig. 5, hei co esponding s o age moduli G 0 a a shea s ess s = 0.05 Pa a e plo ed agains he equency (Fig. 6).All s o age moduli a e independen om he used equency indica - ing e e y emulsion o ha e gel-like p ope ies. Ob iously he e a e big di e ences in he absolu e alues o G 0 . In e es ingly, he s o age modulus G 0 inc eased om 100 Pa (use o HPB alone) o mo e han 2000 Pa due o syne gis ic use o HPB and boehmi e. I he s o age moduli would be de e mined by he num- be densi y o he oil d ople s, bo h s o age moduli o he emul- sions p epa ed wi h HPB a e simply much oo high. I had been concluded he e o e ha he s o age modulus o he p o ein emul- sions a e due o he elas ici y o he p o ein ne wo k ha is o med by he in e ac ing p o ein monolaye s a ound he oil d ople s [13,21] As he boehmi e pa icles a e co e ed by he p o ein, i is likely ha he in e ac ion in HPB based emulsions is e y simila , because in each case he con ac be ween he emulsion d ople s is a p o ein–p o ein con ac . The igid, needle-like boehmi e pa i- cles a e ob iously ac ing as s i ene o he sel -suppo ing, h ee- dimensional hyd ophobin ne wo k. Viscoelas ic ne wo ks in gen- e al show a big di e si y in he needed de o ma ion o induce hei b eakdown [25,26]. Deepe heological in es iga ions showed ha addi ion o boehmi e inc eases he ole ance agains de o ma ion o emulsions based on HPB om 10% o almos 20%. The emulsion p epa ed om hyd ophobin coa ed boehmi e pa - icles (Fig. 5c) was also in es iga ed wi h he C yo-SEM echnique (Fig. 7). Fig. 7a p o ides an o e iew o he emulsion d ople s uc u e. Due o he almos high in e nal oil phase con en , he d ople s a e closed packed and ouch each o he . This spa ial limi a ion will also suppo he en anglemen o he hyd ophobin–boehmi e nanopa icles coa ing he emulsion d ople s esul ing in he o ma- ion o he igid ne wo k. Fu he mo e he emulsion d ople s a e o a ound shape and hey seem o be ha dly polydispe se. Thei size is in good ag eemen as well as wi h heo e ical calcula ions using he co e–shell model as wi h esul om ligh mic oscopy in es i- ga ions. The a e age emulsion d ople size using ligh mic oscopy was de e mined as 1.05 ± 0.24 l m. In Fig. 7b some o he emulsion d ople s a e co e ed wi h b igh spo s ha a e o ming s uc u es (whi e a ows). F om hei size hey could be ega ded as he hyd ophobin coa ed boehmi e pa i- cles. A ew such spo s a e also isible in he aqueous bulk laye . Fig. 2. Mix u es p epa ed om 1 w .% boehmi e and 1 w .% H S a P o ein Ò B (HPP) a pH 6. Fig. 3. Su ace ension o he supe na an s o samples wi h 0.5 w .% boehmi e and inc easing concen a ions o HPB in compa ison wi h he su ace ension o HPB alone. D op- o ma ion speed was 3 s/ l l. Table 2 Amoun s o adso bed and ee hyd ophobin (HPB) in w .% o samples a a fixed boehmi e concen a ion o 0.5 w .%. HPB o al (w .%) 0.03 0.05 0.08 0.1 0.25 0.5 0.75 1 1.5 2 2.5 3 HPB ee (w .%) 0 0 0 0 0.08 0.2 0.4 0.55 0.9 1.5 2 2.5 HPB adso bed (w .%) 0.03 0.05 0.08 0.1 0.17 0.3 0.35 0.45 0.6 0.5 0.5 0.5 4M. Rege , H. Ho mann / Jou nal o Colloid and In e ace Science xxx (2011) xxx–xxx Please ci e his a icle in p ess as: M. Rege , H. Ho mann, J. Colloid In e ace Sci. (2011), doi:10.1016/j.jcis.2011.10.050 70 In e es ingly, he sample wi hou boehmi e did no show any sign o hese spo s. Ne e heless, hei numbe is qui e low and hei dis ibu ion among he emulsion d ople s is no homogenous. I is he e o e no possible o say defini ely ha hese spo s a e he modified boehmi e pa icles. Un o una ely, i was no possible o ob ain C yo-SEM mic og aphs a highe magnifica ions as he sample end o s ong cha ging e ec s. I is also impo an o in es iga e he influence o he used emulsifica ion machine, espec i ely o exe and high p essu e emulsifie . The emulsion d ople size depends ypically on he shea s ess used in he emulsifica ion p ocess [27]. The e o e, he emul- sion d ople size and he s o age moduli G 0 in dependency o he used shea s ess o a Picke ing emulsion p epa ed wi h he same amoun s o emulsifie , wa e and oil was in es iga ed (Table 3). Acco ding o Table 3 wi h inc easing shea s ess he emulsion d op- le size as well as he d ople s polydispe si y dec eased. The bigges change was ob ained by swi ching om Vo ex shake o he high p essu e emulsifie a 100 ba . A second e ec can be obse ed by moni o ing he s o age moduli G 0 in dependency o inc easing shea s ess. The beha io o G 0 is con a y o i o he emulsion d ople Fig. 4. C yo-TEM mic og aphs o 0.5 w .% boehmi e and o a mix u e o 0.5 w .% boehmi e and 0.5 w .% HPB a di e en magnifica ions. Fig. 5. Demons a ion o syne gis ic emulsi ying ac ion. Shown a e high p essu e (1000 ba ), 1 day old emulsions p epa ed om 0.5 w .% HPB (a), 0.5 w .% boehmi e (b) and 0.5 w .% HPB/0.5 w .% boehmi e (c) as emulsifie s, oil mass ac ion U = 0.65 PDMS. Fig. 6. T end o s o age moduli G 0 (Pa) o emulsion p epa ed om 0.5 w .% boehmi e (open ci cles), 0.5 w .% HPB (closed ci cles) and 0.5 w .% HPB and 0.5 w .% boehmi e (hal -closed ci cles) ac ing as emulsifie . The da a we e ob ained 1 day a e emulsifica ion a a shea s ess s o 0.05 Pa. M. Rege , H. Ho mann / Jou nal o Colloid and In e ace Science xxx (2011) xxx–xxx 5 Please ci e his a icle in p ess as: M. Rege , H. Ho mann, J. Colloid In e ace Sci. (2011), doi:10.1016/j.jcis.2011.10.050 71 size. Wi h using highe shea s esses, G 0 inc eases, oo. I is ob ious, ha a smalle emulsion d ople size co esponds o a highe numbe o oil d ople s. This a ec s he o ma ion and s i ness o he sel suppo ing, h ee-dimensional hyd ophobin–boehmi e ne wo k. The enla ged numbe o oil d ople s can fill up he ne wo k mo e densely. Mo eo e i p omo es he s i ness o he ne wo k by c ea - ing mo e ie poin s o p o ein–p o ein en anglemen be ween indi- idual emulsion d ople s. In emulsion science and a he in i s applica ion in indus ial p oduc s, like cosme ic c eams, he e is a g ea in e es o ob ain a long- e m s able, homogenous emulsion laye . The e o e, i is indispensable o in es iga e he hyd ophobin coa ed boehmi e based emulsion o ypical emulsion ins abili y p ocesses like c eaming, Os wald Ripening o d ople coalescence [28–30]. Typical ways o ollow emulsion s abili y include moni o ing he emulsion d ople size and he emulsions’ isual appea ance in dependency o ime. I is no ewo hy ha he homogenous, single laye Picke ing emulsions p epa ed wi h he high p essu e emulsi- fie (Table 4) did no show any isual sign o ins abili y, like oil c eaming o phase sepa a ion, e en a e 170 days s o age a oom empe a u e. Mo eo e he emulsion d ople size did no change a all (Table 4), indica ing he Picke ing emulsions o ha e long- e m du abili y. Ou o me in es iga ions [13,21] p o ed al eady ha emul- sions based on hyd ophobin show ageing e ec s moni o ed by he s o age moduli G 0 in dependence o ime. We obse ed simila e ec s in HPB coa ed boehmi e based emulsions (Fig. 8). The Pick- e ing emulsions show a s ong ipening wi hin he fi s day a e p epa a ion indica ed by he doubling o he G 0 alues. The aging in hese is due o he e olu ion o a h ee-dimensional ne wo k o he c oss-linked hyd ophobin–boehmi e co e ed d ople s while he size o he d ople s does no change wi h ime. Changes in he con o ma ion o he p o ein molecules and o a pa ial en angle- men o p o ein molecules wi h each o he among he whole emul- sion laye a e likely o be esponsible o he ageing phenomena. The s icky hyd ophobin coa ed boehmi e adso bed a he o/w in e ace can bind o each o he by hyd ogen-bonding, an-de Waals a ac ion o he o ma ion o chemical bonds by cys eine esidues. The s ong endency o sel -agg ega ion o hyd ophobins was p o en in ou o me s udy, showing he su ace ension o HPB is s ongly ime dependen indica ing some molecula ea - angemen s o ake place. Indeed, he p esence o ou disulfide b idges make HPB uly a igid molecule [10], bu ob iously wi h- ou loosing i s flexibili y o unde go adso p ion induced molecula eo ganiza ions and in e ac ions. As lysozyme also has ou disul- fide b idges and is p o en o unde go simila e olding p ocesses a e adso p ion, i is a good compa ison o HPB [31]. In e es ingly, howe e , is he ac ha he s o age moduli quad upled a e 170 days (Fig. 8). As he samples ha e been closed igh ly, no wa e should ha e been e apo a ed. I seems ha he final o ma ion o he ne wo k is a long- ime p ocess. As he hyd ophobins con ain ou conse ed disulfide b idges, i was in e es ing o no e he influence o he addi ion o a ypical disulfide b eake like di hio h ei ol (DTT) on he Picke ing emul- sion. As no significan changes in he emulsion appea ance as well as in i s heological p ope ies could be obse ed, we assume he disulfide bonds no o be in ol ed in he o ma ion o he sel - suppo ing p o ein ne wo k. 3.4. E ec s o pa ame e a ia ion o he emulsions In his sec ion we wan o s udy he influence o a ying di e - en pa ame e s o he Picke ing emulsions based on hyd ophobin coa ed boehmi e pa icles. An impo an poin is he in es iga ion o he oil mass ac ion dependence on he emulsion homogenei y and elas ic p ope ies. We assume he gel-like p ope ies o he emulsions esul om he a ac i e in e ac ion be ween he hyd ophobin–boehmi e co e ed oil d ople s. The dense laye o he p o ein co e ed pa icles on he oil d ople s will y o inc ease hei con ac s, un il he epulsi e o ces o igina ing om he pack- ing will o e come he a ac i e o ces. The e o e dilu ed, eshly p epa ed, homogenously looking emulsion laye s will con ac , un il bo h o ces a e balanced. The esul will be a wo laye si u- a ion, espec i ely in ou case an uppe emulsion laye and a lowe aqueous phase. Expe imen s wi h a ying he PDMS oil mass Fig. 7. C yo-SEM in es iga ion o he Picke ing emulsion con aining 0.5 w .% HPB, 0.5 w .% boehmi e and an oil mass ac ion U o 0.65 PDMS p epa ed a 1000 ba . Whi e a ows in (b) show s uc u es ha could be o med by he hyd ophobin coa ed nanopa icles. Table 3 A e age emulsion d ople size ( l m) and s o age moduli G0(Pa) in dependency o he emulsifica ion me hod, espec i ely Vo ex shake and high p essu e emulsifie . The emulsion con ained 0.5 w .% HPB, 0.5 w .% boehmi e and an oil mass ac ion U o 0.65 PDMS and was in es iga ed di ec ly a e p epa a ion. Vo ex shake High p essu e emulsifie 100 ba 300 ba 1000 ba G 0 (Pa) a s = 0.5 Pa and = 1 Hz 12.8 761 1166 1352 A e age d ople size ( l m) 17.67 ± 14.40 1.39 ± 0.47 1.19 ± 0.24 1.05 ± 0.24 6M. Rege , H. Ho mann / Jou nal o Colloid and In e ace Science xxx (2011) xxx–xxx Please ci e his a icle in p ess as: M. Rege , H. Ho mann, J. Colloid In e ace Sci. (2011), doi:10.1016/j.jcis.2011.10.050 72 ac ion U we e pe o med in o de o de e mine he minimum PDMS mass ac ion o ob aining a single laye emulsion. All emul- sion con ained 0.5 w .% HPB, 0.5 w .% boehmi e and ha e been p e- pa ed wi h he high p essu e emulsifie a 300 ba . Wi h using oil mass ac ion U lowe han 0.3, he homogenous emulsion laye con ac wi hin a sho ime, esul ing in a wo laye sys em. Wi h inc easing he oil mass ac ion, he p og ess o he emulsion laye ’s con ac ion was weake . Long- e m s able, homogenous Picke ing emulsions need o ha e an oil mass ac ion U highe han 0.3. The end o he s o age moduli G 0 in dependency o he used oil mass ac ion U is shown in Fig. 9. In o de o be ee om aging e ec s, he heological measu emen s we e immedia ely pe o med a e he emulsifica ion p ocess. The s o age moduli G 0 is inc easing wi h highe oil mass ac- ions U , un il almos a G 0 -pla eau a U P0.5 is eached. A hese oil mass ac ions he eshly o med, loose h ee-dimensional ne - wo k o he hyd ophobin co e ed boehmi e is almos filled. A u - he inc ease in he s o age moduli due o aging e ec s will he e o e be de ec ed wi h longe incuba ion imes as shown in Fig. 8. Fo possible indus ial o cosme ic applica ions, i is also o g ea in e es o check wha ype o oils can be emulsified by he sys em. In o de o in es iga e homogenous emulsion laye s, Pick- e ing emulsions based on 0.5 w .% HPB, 0.5 w .% boehmi e and an oil mass ac ion U o 0.65 ha e been p epa ed wi h he high p es- su e emulsifie a 300 ba . The chosen oils we e Dodecane, an apola and low molecula weigh oil, he silicon oil PDMS and he pola oil Oc yl-me hoxycinnama e (OMC). Using ou sys em enabled o emulsi y all h ee ypes o oil. By eye all emulsions looked qui e simila : They we e homogenous single laye emul- sions wi h oo hpas e like appea ance and a yield s ess alue ha p e en s he emulsions om o ming a ho izon al meniscus. In Ta- ble 5 he a e age d ople size and he s o age moduli G 0 ( s = 0.5 Pa; = 1 Hz) o each oil ype is summa ized. The a e age d ople sizes as well as he s o age moduli o he h ee oils do no di e significan ly (Table 5). The s o age modulus o an emulsion is a pa ame e ha can a y many o de s o magni- ude o emulsions wi h he same s uc u e and he same dimen- sion o he d ople s (2 l m). As he oil ype ob iously does no influence he s o age moduli d ama ically, ou assump ion ha he s o age modulus o he emulsions is mainly con olled by he elas ic p ope ies o he films a ound he oil d ople s is again confi med. Finally, i is also e y impo an o de e mine he minimum con- cen a ion o he hyd ophobin coa ed boehmi e pa icles ha a e needed o main ain he abili y o emulsi y high mass ac ions o oil. Emulsifica ion expe imen s we e pe o med wi h keeping he a io o hyd ophobin o boehmi e 1:1, bu lowe ing hei o al con- cen a ion. The ob ained samples a e shown in Fig. 10. The emul- sions con aining 0.05 and 0.1 w .% o each, HPB and boehmi e, a e uns able, mul ilaye sys ems. Some oil has immedia ely c eamed up, he emulsion laye i sel is inhomogeneous. The emulsion p e- pa ed wi h 0.25 w .%, howe e , is homogenous and shows signs o he al eady obse ed oo h-pas e cha ac e o emulsion con aining 0.5 w .% o HPB and boehmi e (Fig. 5). E en he yield s ess is significan ly highe , as his emulsion does no o m a ho izon al meniscus. I has o be epo ed ha he appea ance o he emulsions con- aining 0.05 and 0.1 w .% HPB and boehmi e showed a big shea a e dependency. I was possible o emulsi y 65 w .% PDMS using he Homo Dispe a low shea a es o 1000 pm. Applying hese emulsions o he high p essu e emulsifie , howe e , lead o pa ial b eakdown o i s emulsi ying abili ies (Fig. 10). This phenomenon can be explained as ollows: The highe he shea s ess he mo e he oil d ople size dec eases and he mo e he eshly p oduced in e ace inc eases esul ing in a much highe oil d ople numbe . Table 4 A e age emulsion d ople size ( l m) in dependency o s o age ime a 25 °C o Picke ing emulsions p epa ed wi h he high p essu e emulsifie . The emulsions con ained 0.5 w .% HPB, 0.5 w .% boehmi e and an oil mass ac ion U o 0.65 PDMS. S o age ime (d) a 25 °C 100 ba 300 ba 1000 ba 0 1.39 ± 0.47 1.19 ± 0.24 1.05 ± 0.24 7 1.40 ± 0.43 1.19 ± 0.24 1.05 ± 0.29 170 1.43 ± 0.34 1.21 ± 0.21 1.08 ± 0.28 Fig. 8. T end o s o age moduli G 0 (Pa) ( s = 0.5 Pa; = 1 Hz) in dependency o s o age ime o high p essu e emulsions. The emulsions con ained 0.5 w .% HPB, 0.5 w .% boehmi e and an oil mass ac ion U o 0.65 PDMS. Fig. 9. T end o s o age moduli G 0 (Pa) ( s = 0.5 Pa; = 1 Hz) in dependency o he used oil mass ac ion U o high p essu e emulsion measu ed di ec ly a e p epa a ion. The emulsions con ained 0.5 w .% HPB, 0.5 w .% boehmi e and an oil mass ac ion o 0.65 PDMS. Table 5 A e age d ople size ( l m) and s o age moduli G0(Pa) ( s = 0.5 Pa; = 1 Hz) o Picke ing emulsions p epa ed wi h di e en ypes o oil, espec i ely dodecane, PDMS and Oc yl- me hoxycinnama e (OMC). All emulsions we e p epa ed om 0.5 w .% HPB, 0.5 w .% boehmi e and an oil mass ac ion U o 0.65 a 300 ba . The in es iga ions we e di ec ly a e emulsifica ion pe o med. Oil ype Dodecane PDMS OMC A e age d ople size ( l m) 1.51 ± 0.51 1.19 ± 0.24 1.27 ± 0.31 G 0 (Pa) ( s = 0.5 Pa; = 1 Hz) 1718 1166 1707 M. Rege , H. Ho mann / Jou nal o Colloid and In e ace Science xxx (2011) xxx–xxx 7 Please ci e his a icle in p ess as: M. Rege , H. Ho mann, J. Colloid In e ace Sci. (2011), doi:10.1016/j.jcis.2011.10.050 73 o he p o eins beside hyd ophobin ha e simila p ope ies as he ones p epa ed wi h hyd ophobin coa ed clay pa icles [17]. Ma e ials and Me hods H S a P o ein ® B, abb e ia ed as HPB, is a ecombinan hyd ophobin15 and was p o ided by BASF SE, Ludwigsha en. HPB has a molecula weigh o 19 kDa and an isoelec ic poin (IEP) o 6.15. HPB consis s o he class I hyd ophobin DewA om he ungi Aspe gillus nidulans and a unca ed o m o he Bacillus sub ilis p o ein yaaD. Lyophilized bo ine se um albumin (BSA) was pu chased om Se a Elec opho esis, Heidelbe g. The molecula weigh o BSA is a ound 67 kDa and has a 4.7 i s IEP. Plan asol W is a hyd olyzed whea p o ein p oduced by Geli a GmbH, Ebe bach. The p o ein con en is gi en as 77-85 w % wi h an a e age molecula weigh o 1-5 kDa wi h an IEP o 4 o 4.5. Nu i ionRx, Buchholz, supplied he soy p o ein isola e wi h an a e age p o ein con en o 87 w %. The yeas ex ac was bough om Ca l Ro h GmbH & Co KG, Ka ls uhe. The p o ein con en is epo ed o be a ound 70 w %. One i h o he d y subs ance o Saccha omyces ce e isiae is e e ed o be o igina ed om he cell wall. A majo componen o i is he mannop o ein (50%), which has long been known as e ec i e bioemulsi ie 31. As Plan asol W, soy p o ein isola e and yeas ex ac a e quie cheap ma e ials, we jus used hem as ecei ed. The clay Laponi e XLG32 was bough om Rockwood Clay Addi i es GmbH, Moosbu g. Polydime hylsiloxane (PDMS) wi h a gene al o mula ion o (CH3)3SiO[(CH3)2SiO]nSi(CH3)3 was pu chased om Shine su Kagaku, Tokyo. I s polyme iza ion deg ee η is anging om 5 o 19 (>98 %) and he iscosi y is app oxima ely 6 mPas. O he chemicals no speci ied in he ex we e o analy ical g ade o equi alen . Wi h he olume-d op ensiome e TVT1 om Lauda Co., Königsho en, he su ace ension σ o he samples was measu ed a a cons an d op- o ma ion speed o 1 µl/s. In o de o de e mine he ee amoun o p o ein in he clay-p o ein mix u es he supe na an o he samples was used. The e o e he samples we e cen i uged in a Medi uge om He aeus Ins umen s GmbH, Hanau, o 10 minu es a 2000 g. All emulsions we e p epa ed om aqueous solu ions o p o eins alone o in combina ion wi h clay. All emulsions con ained 0.5 w % phenoxye hanole in o de o supp ess mic obial g ow h. As one s ep oil addi ion o he aqueous phase led o emulsion b eakdown, high oil con en emulsions could only be ob ained by s epwise addi ion o oil. Be o e using he High P essu e Emulsi ie (APV 1000, Albe slund) he emulsions we e p e-emulsi ied wi h he Homo Dispe (Tokushu Kika, Osaka) a an emulsi ica ion speed o abou 1000 e olu ions pe 80 minu e ( pm). The p e-emulsions we e ans e ed o he High P essu e Emulsi ie and passed h ee imes h ough he de ice a he desi ed p essu e o 300 ba . Fo Ligh Mic oscopy in es iga ions ollowing p o ocol was used. As mos o he emulsions ha e a gel-like beha iou due o he s icky cha ac e o he emulsion d ople s, i was no possible o dilu e he samples di ec ly wi h wa e . The e o e ano he way had o be chosen. A e applying he emulsion laye o he mic oscope slide and closing i wi h he co e slide, i was jus gen ly shi ed a bi , in o de o ge a hin emulsion laye . Then ocusing h ough he sample ne e esol ed mo e han wo d ople laye s a he same ime. Consequen ly, using one sha pness le el ga e a good o e iew abou he a e age d ople size. Mo eo e he polydispe si y was low and indica es he ligh mic oscopy o be app op ia e in o de o de e mine he a e age d ople size. The ligh mic oscope was om Zeiss, espec i ely he model: 47 60 05 – 9901. Using he DFK 41F02 came a allowed o digi alize he mic og aphs. The analysis was pe o med wi h he IC cap u e 2.1 so wa e (The Imaging Sou ce, B emen). The heology o he emulsion laye s was measu ed a 25 °C wi h a cone-pla e heome e RheoS ess 600 om Haake The mo Scien i ic, Ka ls uhe. The Haake RheoWin Da a Manage , Ve sion 3.3, was used o analyze he expe imen al da a. Resul s and Discussion Su ace ension measu emen s P o eins a e ampho e ic molecules and he e o e lowe he su ace ension o aqueous solu ions [33;34]. This p ope y is p o ided by he building blocks ha p o eins a e made om. In o de o cha ac e ize he su ace ac i i y o he i e p o eins used in his s udy, we p esen he e hei su ace ension p o ile in he concen a ion ange om 0.01 o 5 w % (Fig. 1). The d op o ma ion speed was adjus ed o 1 s/µl. 81 Fig. 1 Su ace ension p o ile o HPB (closed ci cles), Soy Isola e (open ci cles), Yeas ex ac (closed squa es), Whea P o ein-Plan asol W (open squa es) and BSA (s a s). The su ace ension was measu ed a a d op o ma ion speed o 1 s/µl. All used p o eins lowe he su ace ension o aqueous solu ions. The su ace ension dec easing abili y inc eases om yeas ex ac o e BSA, Plan asol W (whea p o ein) and soy p o ein isola e o HPB. HPB is he e o e he mos su ace ac i e p o ein in ou s udy. In li e a u e hyd ophobins a e e e ed o be he mos su ace ac i e p o eins o be known a he momen [35,36]. The de ailed expe imen al su ace ension alues o concen a ion o 0.5 w % and 5 w % a e summa ized in ab. 1. Tab. 1 O e iew o he su ace ension σ o i e di e en p o eins a concen a ions o 0.5 and 5 w %. The d op o ma ion speed was adjus ed a 1 s/µl. yeas ex ac BSA Plan asol W soy p o ein isola e HPB 0.5 w % 72.86 ± 0.04 mN/m 66.39 ± 0.03 mN/m 61.25 ± 0.08 mN/m 57.56 ± 0.05 mN/m 50.31 ± 0.06 mN/m 5 w % 63.52 ± 0.02 mN/m 60.91 ± 0.60 mN/m 53.28 ± 0.22 mN/m 45.49 ± 0.18 mN/m 42.57 ± 0.10 mN/m I should be no ed ha p elimina y s udies showed ha aqueous solu ions o soy p o ein isola e a e wo laye sys ems. The swollen, u bid lowe phase makes app oxima ely 30% o he o al sample olume. We ied o o e come his wo phase sepa a ion by changing he pH o lowe (+HCl) and highe (+NaOH) alues. I u ned ou ha a a pH o 12 he sample became homogenous. Su ace ension measu emen s howe e poin ed ou , ha σ o 1 w % solu ions o soy p o ein isola e inc eased om 51.84 ± 0.07 (pH 6) o 55.69 ± 0.36 (pH 12). As we assume he emulsion p ope ies o be di ec ly linked o he su ace ac i i y, we used o he emulsion p epa a ion he soy p o ein isola e in he wo laye s a e. Emulsions p epa ed om di e en p o eins In his sec ion we wan o compa e he isual p ope ies, s abili y, emulsion d ople size and heological beha iou o emulsions based on he p o eins used in his s udy. The e o e we kep he condi ions simila o all emulsions, bu jus changed he p o ein ype. Fo he 82 emulsi ica ion p ocess he high p essu e emulsi ie was used a 300 ba . An emulsi ie concen a ion o 0.5 w % was chosen, he oil con en was adjus ed o 50 w % polydime hylsiloxane (PDMS). In ig. 2 he ob ained emulsions a e illus a ed di ec ly a e p epa a ion (uppe ow) as well as h ee days a e incuba ion a oom empe a u e (lowe ow). Conduc i i y measu emen s indica ed all emulsions o be o he oil in wa e ype. Immedia ely a e p epa a ion he emulsions based on Plan asol W and yeas ex ac s a o phase sepa a e. A e 3 days he emulsion laye disappea ed, an uppe oily and a lowe aqueous phase emained. Al hough he oil mass ac ion should be 0.5, hese wo emulsions do no show equal ac ions o oil and wa e (lowe ow). This is p obably a consequence o he ac ha bo h p o eins ha e weak emulsi ying abili ies. While illing he high p essu e emulsi ie wi h he p e-emulsion, i b oke almos comple ely up. Consequen ly due o he lowe densi y o PDMS, i mo ed upwa ds in he solu ion. A di ec mix u e o oil and wa e in he emulsi ica ion machine was no possible anymo e. The wo bo led emulsions he e o e showed no equal olume a ios o oil and wa e . Fig. 2 Emulsions con aining 50 w % PDMS s abilized by 0.5 w % o di e en p o eins. The uppe ow shows he emulsion di ec ly a e p epa a ion, while he lowe ow demons a es he si ua ion a e 3d incuba ion a oom empe a u e. The do ed line in he Plan asol W based emulsion is as guide o he eye in o de o demons a e he phase bounda y. The illing le el is di e en o echnical easons. Somewha di e en is he si ua ion o he emulsions based on soy p o ein isola e, BSA and HPB. Di ec ly a e p epa a ion all o hem a e homogenous emulsions. A e h ee days incuba ion a oom empe a u e, howe e , he BSA and HPB based emulsions ha e phase sepa a ed indica ed by he appea ance o lowe wa e like phases. Mo eo e he BSA based emulsion p o ided an addi ional ins abili y p ocess, known as oil c eaming. Con a y o all 83 o he emulsions he one based on soy p o ein isola e was s ill homogenous and did no p esen any signs o ins abili y. As no ed be o e, emulsion des abilisa ion mechanisms include p ocesses ha a e known as coalescence o Os wald-Ripening. These changes in he emulsion s uc u e a e accompanied by an inc ease in he a e age emulsion d ople size (ads). In o de o check he s udied emulsions o d ople size al e ing, hei a e age d ople size was de e mined di ec ly a e p epa a ion and h ee days la e by ligh mic oscopy ( ab. 2). Tab. 2 O e iew o he a e age d ople size (ads) o emulsions based on di e en p o eins. The size was e alua ed di ec ly a e p epa a ion and h ee days la e by ligh mic oscopy. The emulsion con ained 0.5 w % p o ein and an oil mass ac ion Φ o 0.5 PDMS. yeas ex ac BSA Plan asol W soy p o ein isola e HPB 0 days no de e minable 2.6 ± 1.2 2.0 ± 0.7 2.6 ± 0.5 2.0 ± 0.9 3 days no de e minable 3.7 ± 3.8 no de e minable 2.8 ± 1.1 1.9 ± 1.0 The a e age emulsion d ople size could no be de e mined in he case o he emulsion based on 0.5 w % yeas ex ac as he emulsion immedia ely b oke. E en he ads wi h 2.0 ± 0.7 o he Plan asol W emulsion migh be much oo low o he eal one as spon aneous coalescence was obse ed. Ne e heless he ads (0 days) in he case o BSA, HPB and soy p o ein isola e seemed o be e y simila and in he ange o heo e ical calcula ions using he co e shell model (see suppo ing in o ma ion). Mo eo e he polydispe si y o he d ople s is qui e small. A e 3 days he ads and mainly he polydispe si y inc eased in he case o BSA, while hey s ayed almos cons an o HPB and soy p o ein isola e. We conclude ha he emulsions p epa ed om HPB and soy p o ein isola e a e s able, maybe e en o longe incuba ion imes. This was al eady p o en in he case o HPB [16]. In e es ingly he only emulsion ha s ayed homogenous unde he used condi ions was ob ained om soy p o ein isola e. 84 Fig. 3 T end o he s o age moduli G’ [Pa] a τ = 0.5 Pa o emulsions based on 0.5 w % o i e di e en p o eins. The s o age moduli o he emulsion laye s we e e alua ed di ec ly a e emulsi ica ion (abo e) and h ee days la e (down). The emulsion con ained 0.5 w % p o ein and an oil mass ac ion Φ o 0.5 PDMS. In he case o emulsions p epa ed om HPB, we concluded he HPB co e ed emulsion d ople s ac as s icky pa icles. Thei in e ac ion esul ed in he o ma ion o a h ee- dimensional ne wo k [16]. Acco dingly he s o age moduli G’ o hese emulsions was much highe han i would be, i i was de e mined by he olume ac ion o he d ople s and hei size dis ibu ion (see suppo ing in o ma ion). The ques ion a ises, i simila e ec s can be obse ed in he emulsions om ig. 2. The e o e he heog ams a a shea s ess τ = 0.5 Pa we e measu ed. Fig. 3 shows he end o he s o age moduli G’ o he i e emulsion sys ems used in his s udy di ec ly a e emulsi ica ion and h ee days la e . F om he equency independen beha iou o G’ in ig. 3 o he emulsion based on HPB and soy p o ein isola e one can di ec ly conclude ha hese emulsions ha e elas ic p ope ies di ec ly a e p epa a ion. Mo eo e he gel-like cha ac e inc eased ema kable wi h u he incuba ion ime. Whe eas he emulsions based on BSA, Plan asol W and yeas ex ac show clea ly iscous beha iou as he s o age moduli G’ is equency-dependen . In e es ingly he bigges change in he s o age moduli G’ seemed o be obse ed o he emulsion based on BSA, espec i ely om iscous o s ong elas ic appea ance. I u ned ou , howe e , ha he oil was squeezed ou o he emulsion while measu ing he heog am a e 3 days ( ig. 4). The emulsion was agglome a ed and s icky, bu no longe homogenous. 85 Fig. 4 Visual appea ance o he emulsion based on 0.5 w % BSA and an oil mass ac ion Φ o 0.5 PDMS a e heological in es iga ions. F om he ob ained esul s in his chap e , we claim he unmodi ied soy p o ein isola e o ha e he bes emulsi ica ion abili ies unde he used condi ions o all p o eins examined in his s udy. Emulsions p epa ed om clays co e ed wi h di e en p o eins As men ioned al eady in he in oduc ion pa , ecen ly we epo ed a no el hyd ophobin clay syne gism esul ing in oo h-pas e like emulsions [17]. Homogenous emulsions could be ob ained wi h an oil mass ac ion o mo e han 0.3. In o de o check i such emulsions can also be p epa ed om o he p o eins han HPB, we eplaced i wi h Plan asol W, soy p o ein isola e, BSA and yeas ex ac . All emaining condi ions we e kep he same; e e y emulsion con ained 0.5 w % p o ein and an oil mass ac ion Φ o 0.5 PDMS. The used p essu e was 300 ba . The isual appea ance o hese o/w emulsions di ec ly a e p epa a ion and h ee days la e is shown in ig. 5. The i s no ewo hy hing is ha he emulsi ying abili y o Plan asol W and yeas ex ac boos ed in compa ison o he si ua ion in ig. 2. Mo eo e o each p o ein-clay combina ion he co esponding emulsion was homogenous di ec ly a e p epa a ion ( ig. 5; uppe ow). A ce ain ipening o he emulsion laye a e h ee days incuba ion a oom empe a u e, howe e , was obse ed o all emulsions beside he one p epa ed wi h he hyd ophobin HPB ( ig. 5,; lowe ow). Fu he mo e his emulsion did clea ly no o m a ho izon al meniscus indica ing i s gel-like p ope ies can al eady be ecognized by eye. 86 Fig. 5 Emulsions con aining 50 w % PDMS and s abilized by 0.5 w % o di e en p o eins and 0.5 w % Laponi e XLG. The uppe ow shows he emulsion di ec ly a e p epa a ion, while he lowe ow demons a es he si ua ion a e 3d incuba ion a oom empe a u e. As done in he case o he emulsions p epa ed om he p o eins alone, he a e age d ople size (ads) was also measu ed in dependency o incuba ion ime o he emulsions ( ab. 3). The ads s ayed cons an i BSA, soy p o ein isola e o HPB was used in combina ion wi h clay. In e es ingly he wo p o eins ha o m uns able emulsions, did also p o ide an inc ease in he ads as well in he d ople polydispe si y in dependency o ime. Coalescence as ypical emulsion des abiliza ion mechanism was he e o e likely o ha e happened. Tab. 3 O e iew o he a e age d ople size (ads) o emulsions based on di e en p o ein-clay combina ions. The size was e alua ed di ec ly a e p epa a ion and h ee days la e by ligh mic oscopy. The emulsion con ained 0.5 w % p o ein, 0.5 w % Laponi e XLG and an oil mass ac ion Φ o 0.5 PDMS. yeas ex ac BSA Plan asol W soy p o ein isola e HPB 0 days 5.1 ± 0.8 1.4 ± 0.4 5.3 ± 1.1 2.5 ± 0.7 1.9 ± 0.4 3 days 8.9 ± 3.1 1.3 ± 0.3 8.5 ± 2.2 2.7 ± 0.8 1.8 ± 0.4 Fo emulsions p epa ed om HPB, he addi ional use o clay esul ed in an inc ease o he gel-like p ope ies o he co esponding emulsions. The s o age modulus G’ ose o alues en imes highe han wi hou clay [17]. As he concen a ion o he clay was wi h 0.5 w % a om i s sol-gel ansi ion, we concluded ha he hyd ophobin co e ed clay pa icles, ac as s icky sandwiches. The clay in oduced he e o e an addi ional ein o cemen o he sel - suppo ing, h ee-dimensional hyd ophobin ne wo k ha showed an inc eased s i ness wi h 87 longe incuba ion imes. In ig. 6 he end o he s o age moduli G’ o he i e emulsion sys ems ( ig. 5) used in his s udy di ec ly a e emulsi ica ion and h ee days la e is gi en. Fig. 6 T end o he s o age moduli G’ [Pa] (τ= 0.5Pa) o he emulsions laye s p epa ed om di e en p o ein- clay mix u es in dependency o ime (0 days: uppe ow; 3 days: lowe ow). All emulsions we e p epa ed om 0.5 w % p o ein, 0.5 w % Laponi e XLG and an oil mass ac ion Φ o 0.5 PDMS a a p essu e o 300 ba . Re e ing o he uppe ow o ig. 6 he s o age moduli o he emulsions based on HPB o soy p o ein in combina ion wi h Laponi e we e equency independen di ec ly a e p epa a ion. In con as he o he emulsions did no show any signs o gel-like beha iou as hei s o age moduli we e qui e equency-dependen . In e es ingly, e en he emulsion based on BSA ha appea ed o be e y low gel-like a e h ee days s o age, showed aging e ec s ( ig. 6; lowe ow). The s onges e ec s we e su ely obse ed in he case o he whea p o ein (Plan asol W) and yeas ex ac . These emulsions changed om a a he iscous s a e di ec ly a e p epa a ion o a high elas ic beha iou a e h ee days incuba ion a oom empe a u e. The inc ease o he s o age modulus G’ o all p o ein-clay combina ion wi hou BSA is due o he o ma ion o a ime-dependen , sel -suppo ing p o ein-clay ne wo k su ounding he oil d ople s. The su ace ac i e p o ein co e ed clay pa icles adso b apidly o he eshly p epa ed oil wa e in e ace du ing homogeniza ion. The ea e he p o ein molecules unde go ime-dependen , con o ma ional ea angemen s in o de o in e ac mo e e ec i ely wi h he new oil-wa e en i onmen and in e pene a e wi h each o he [37-38]. The p o ein co e ed clay pa icles ac as s icky sandwiches and o m a ime-dependen , sel suppo ing ne wo k 88 ha s eng hens wi h incuba ion ime. Fo he combina ion o clay and hyd ophobin i was ecen ly shown ha his ne wo k emains a e emo ing he oil and wa e by using eeze d ying [17]. F om he di e en elas ic beha iou , i is ob ious ha he emulsion d ople -d ople in e ac ion was di e en and depending on he used p o ein in combina ion wi h clay. The mos e ec i e in e ac ion was obse ed in he case o HPB. Ne e heless, beside BSA, all o he p o eins showed a ime-dependen e ol ing o hei gel-like p ope ies. I has o be no ed ha in he case o he whea p o ein, he emulsion s a ed o smell somehow suspec . E en hough 0.5 w % phenoxye hanole was used o p e en bac e ial o ungal g ow h, i s amoun was p obably no su icien enough. Howe e we did no wan o inc ease he amoun o i o e he amoun o he used p o ein, because om he chemical poin o iew phenoxye hanole can be conside ed as an amphiphile, oo. In o de o p o e his, a emulsion eplacing p o ein by 0.5 w % phenoxye hanole was p epa ed. The o he pa ame e s we e kep he same. The ob ained emulsion sepa a ed quickly in a wo laye sys em and was low- iscous as judged by i s isual appea ance. Rheological measu emen s con i med he iscous beha iou as he s o age modulus was equency dependen . A a shea s ess τ = 0.5 Pa and a equency = 1 Hz, G’ was abou 0.09 Pa. Mo eo e no sign o aging e ec s we e obse ed. Finally in o de o a i m ha all o he used p o eins bind o he clay pa icles su ace ension measu emen s we e conduc ed. The e o e he su ace ension o he supe na an o he p o ein-clay samples was measu ed. The ob ained alue co esponded o he one o he non- adso bed, ee p o ein concen a ion ( ig. 1). By sub ac ion his concen a ion om he o ally used p o ein concen a ion (0.5 w %), he adso bed p o ein concen a ion could be de e mined. Tab. 4 shows he measu ed su ace ension o he supe na an o clay-p o ein mix u es as well as he concen a ion o adso bed and non-adso bed p o ein. Tab. 4 O e iew o he su ace ension σ o he supe na an s o mix u es o 0.5 w % p o ein and 0.5 w % Laponi e XLG as well as he concen a ion o adso bed and non-adso bed p o ein. yeas ex ac BSA Plan asol W soy isola e HPB σ [mN/m] 73.66 ± 0.05 73.21 ± 0.02 71.31 ± 0.02 71.48 ± 0.05 72.07 ± 0.03 adso bed p o ein [w %] 0.4-0.5 0.5 0.47 0.42 < 0.45 ee p o ein [w %] 0-0.1 0 0.03 0.08 < 0.05 The esul s in ab. 4 clea ly show ha in each case mo e han 80% o he used p o ein concen a ion is adso bed a he clay pa icles. In e es ingly in he case o BSA all p o ein is 89 11. Exe owa D, Go che G, Kola o T, Kh is o K, Le ecke B and Tad os T (2007) In e ac ion o ces in hin liquid ilms s abilized by hyd ophobically modi ied inulin polyme ic su ac an . 2. Emulsion ilms, Langmui , 23: 1711-1715 12. 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G adzielski M and Ho mann H (1994) In luence o cha ges on s uc u e and dynamics o an o/w mic oemulsion. E ec o admixing ionic su ac an s, J Phys Chem, 98, 2613–2623 40. G adzielski M, Ho mann H and Oe e G (1990) Ringing Gels: Thei s uc u e and mac oscopic p ope ies, Colloid Polym Sci, 268, 167-178 98 99 Publica ion E Ma in Rege , Tomoko Sekine and Heinz Ho mann, Picke ing Emulsions s abilized by Amphiphile co e ed Clays, Colloids and Su aces A, 2011, accep ed, Ms. Re . No.: COLSUA-D-11-00925. Picke ing Emulsions s abilized by Amphiphile co e ed Clays Ma in Rege *1 , Tomoko Sekine 2 and Heinz Ho mann 1 1 Uni e si y o Bay eu h, BZKG/BayColl, Go lieb-Keim-S aße 60, 95448 Bay eu h, Ge many; 2 Shiseido Resea ch Cen e , 2-2-1 Hayabuchi, Tsuzuki-ku, Yokohama 224-8558, Japan. * Tel.: +49 921 50736168; E-mail add ess: Ma in.Rege @uni-bay eu h.de A B S T R A C T I is shown ha all wa e soluble amphiphilic compounds like su ac an s, polyme s, block copolyme s and p o eins bind s ongly o clay pa icles in wa e . On sa u a ion, he clays bind up o he mul i old weigh o he addi i es o hei own weigh . P io o sa u a ion some amphiphilic compounds o m p ecipi a es wi h he clays while o he s do no . The soluble clay-amphiphile complexes do no lowe he su ace ension o wa e e en hough hese pa icles mus ob iously be hyd ophobic. I is concluded om hese esul s ha he clays alone a e al eady hyd ophobic pa icles. Solu ions o p ecipi a es o hese amphiphile co e ed clay pa icles a e ideal sys ems o he o ma ion o s able Picke ing emulsions. Depending on he used ype and amoun o amphiphile, he co esponding Picke ing emulsions can ha e ei he iscous o gel-like p ope ies wi h la ge s o age moduli. Fo some combina ions bo h he wa e and he oil can be emo ed om he emulsions wi hou collapse o he h ee dimensional ne wo k o he ilms. Keywo ds: Clay, Hyd ophobin, Picke ing Emulsion, Syne gism, TTMAB 1. In oduc ion Clays a e in e es ing compounds o a wide ange o applica ions [1-3]. The mine als can be ex olia ed o single shee s in aqueous solu ions. In his s a e he hin shee s ha e a hickness o one nm. Because o hei la ge su ace o abou 1000 m 2 /g, he clay pa icles can adso b he mani old weigh as hei own weigh . Clays a e he e o e used as adso be s o many was e p oduc s like su ac an s [4], dyes [5] o hea y me als [6] in he pu i ica ion p ocess o wa e . Clays a e a ailable ei he as na u al o syn he ic p oduc s. Na u al clays a e known unde di e en names like Mon mo illoni e and Saponi e. The diame e o he pla e-like clay-pa icles anges om 20 nm o syn he ic clays o many µm o na u al clays. Because o hei la ge anisome y and possible su ace modi ica ion, clays a e o en used o a ious pu poses, o example as heology modi ie s [7]. The heology o indus ial o mula ions can be changed om shea hinning solu ions o s i gels wi h a ew w % o clays. In ecen imes clays ha e been used o s eng hen he s i ness o polyme s [8] and o imp o e he impe meabili y o gases h ough hin polyme ilms [9]. In combina ion wi h su ac an s, clays can be made hyd ophobic and hese hyd ophobic clay-pa icles can be dispe sed in hyd oca bons [10]. In mix u e wi h non-ionic su ac an s clays ha e also been used o he p epa a ion o Picke ing emulsions [11]. This applica ion b ings up an in e es ing basic ques ion. The su ace o clays is gene ally conside ed o be a hyd ophilic su ace. Dispe sions o clays ha e he same su ace ension as wa e [12]. I is he e o e assumed ha clays a e no su ace ac i e, bu his conclusion may no be comple ely co ec . I is known ha polyme s like polye hylene glycol which a e conside ed o be hyd ophilic bind on clays [13-14]. This esul makes only sense, i he su ace o he clays has somehow hyd ophobic ea u es. Recen ly i was shown ha one o one mix u es o clay- hyd ophobin pa icles s abilize high in e nal oil in wa e (o/w) emulsions by syne gis ic in e ac ion [15]. Only 1 w % o hyd ophobin and clay we e needed o p epa e homogenous emulsions. Howe e , i was ema kable ha bo h, he clay and hyd ophobin p o ein a e nega i ely cha ged. Rheological measu emen s showed ha he Picke ing emulsion om his combina ion has a high s o age modulus G’ (> 1000 Pa) and iscosi y (1 Pas a γ = 100 s -1 ). This manusc ip ocuses on h ee main aspec s. The i s pa will be abou he in e ac ion be ween amphiphilic compounds and clay. The e o e especially he su ace ension beha iou o he amphiphile-clay pa icles will be discussed in gene al e ms. The second pa deals wi h he ques ion, wha kind o one o one mix u es o amphiphile and clay will esul in simila emulsions as ob ained wi h he hyd ophobin, called H S a P o ein B ® [16]. The in luence o he clay co e age wi h amphiphile on he isual appea ance, s abili y and heological beha iou o he emulsion is in es iga ed. As a model sys em we used he ca ionic su ac an C 14 - ime hylammoniumb omid (TTMAB ) wi h Laponi e XLG as clay compound. Finally, in he hi d pa , we disc imina ed ou emulsion sys ems om Picke ing emulsions p epa ed wi h silica pa icles [17]. 2. Ma e ials and Me hods 2.1 Ma e ials The ca ionic su ac an C 16 -T ime hylammoniumb omid (CTAB) was bough om Me ck, Da ms ad , whe eas C 14 - T ime hylammoniumb omid (TTMAB ) was pu chased om Cla ian , Gendo . Se a Elec opho esis GmbH, Heidelbe g, p o ided he anionic su ac an Sodium-Dodecylsul a e (SDS). As non-ionic su ac an we chose alkyl polyoxye hylene glycol monome ype C 12 EO 7.8 om Cla ian , Gendo , and Iso idecyloc ae hylenglycole he (C 13 O 8 ) om Sasol, Hambu g. As zwi e ionic su ac an we used Te adecyldime hylaminoxide (TDMAO) om Cla ian , Gendo , and Dis ea yldiammoniumchlo ide (DSDAC) om 100 Sanyo Chemical Indus ies, Japan. The non-ionic iblock copolyme s Plu onic F38 and F127 we e pu chased om BASF, Ludwigsha en. The copolyme Polyoxye hylene 17 - polyoxypo pylene 4 -dime hyle he (AQ 1704) was om Yuka Sangyo Co, Japan. The ecombinan p o ein hyd ophobin, called H S a P o ein ® B (HPB) [16], was a gi om BASF, Ludwigsha en. The poly inylalcohole (PVA, ade name Poly iol ® LL 2860) was acqui ed om Wacke , Bu ghausen. Poly inylpy olidon (PVP) was bough om Henkel, Düsseldo , and Poly-diallyldime hylammoniumchlo ide (DADMAC) was ob ained om Ald ich, S einheim. The clay Laponi e XLG [18] was pu chased om Rockwood Clay Addi i es GmbH, Moosbu g. I has a ca ionic exchange capaci y (cec) o 65.7 meq/100g Polydime hylsiloxane (PDMS) was acqui ed om Shine su Kagaku, Tokyo. I has gene al o mula ion: (CH 3 ) 3 SiO[(CH 3 )2SiO]nSi(CH 3 ) 3 . The polyme iza ion deg ee η is anging om 5 o 19 (>98 %) and he iscosi y is app oxima ely 6 mPas. O he chemicals no speci ied in he ex we e o analy ical g ade o equi alen . 2.2 Su ace Tension The su ace ension σ o he samples was measu ed wi h he olume-d op ensiome e TVT1 om Lauda Co., Königsho en a a cons an d op- o ma ion speed o 1 s/µl. In o de o de e mine he ee amoun o amphiphile in he clay- amphiphile mix u es, he supe na an o he samples was used. The e o e he samples we e cen i uged in a Medi uge om He aeus Ins umen s GmbH, Hanau, o 10 min a 2000 g. 2.3 Emulsion P epa a ion. All emulsions we e p epa ed om aqueous solu ions o amphiphile and clay. Addi ionally all p o ein emulsions con ained 0.5 w % phenoxye hanole in o de o p e en mic obial g ow h. Using he High P essu e Emulsi ie (APV 1000, Albe slund) equi ed p e-emulsi ica ion o he sample using he Homo Dispe (Tokushu Kika, Japan) a low pm alues o a ound 100. A e wa ds he sample was emulsi ied h ee imes a a p essu e o 300 ba . 2.4 Rheology The heology o he emulsions was measu ed wi h a cone- pla e heome e RheoS ess 600 om Haake The mo Scien i ic, Ka ls uhe, a 25 °C. The expe imen al da a was analyzed wi h he Haake RheoWin Da a Manage , Ve sion 3.3. 3. Resul s and discussions 3.1. Clays and Amphiphiles in wa e 3.1.1 Clays and su ac an s In his segmen we explain he gene al beha iou o su ac an -clay mix u es in ela ion o hei isual appea ance and hei e ec s on su ace ension. The schema ic su ace ension p o ile o a su ac an -clay mix u e is compa ed o he one o a pu e su ac an solu ion in ig. 1. Wi h inc easing su ac an concen a ion, he su ace ension o wa e s a s o dec ease con inuously un il he c i ical micella concen a ion (cmc) o he su ac an is eached. Whe eas he clay-su ac an mix u es main ain he same su ace ension as wa e , un il he su ac an monolaye adso p ion concen a ion (smac) is eached. All added su ac an is adso bed on he clay pa icles. A e eaching he smac, s ill su ac an binds o he clay- su ac an pa icles, bu no comple ely as is ob ious om he now dec easing su ace ension. A he su ac an -clay cmc (scc), he ee su ac an in he solu ion inally eaches he su ace ension o a pu e su ac an solu ion a and abo e i s cmc. The amoun o adso bed su ac an can easily be de e mined by sub ac ion o he cmc om he ssc. Fig. 1 shows also some de ails which a e o special in e es o he o mula ion o Picke ing emulsions. A concen a ion egion o he su ac an exis s in which he clay-su ac an pa icles al eady a e o med, bu he solu ions a e clea and no p ecipi a e is o med in he solu ion. This concen a ion egion o single phase a ea is ma ked as 1 Φ. The adso p ion o he su ac an molecules mus ha e made he su ace o he clays mo e hyd ophobic, bu he emaining ca ionic cha ge o he clays is s ill s ong enough o p e en p ecipi a ion. These could be condi ions in which al eady s able Picke ing emulsions a e o med. Wi h inc easing adso p ion o he su ac an molecules he clay-pa icle become so hyd ophobic ha hey o m a p ecipi a e. This egion is indica ed in Fig. 1 as 2 Φ. The concen a ion o su ac an ha is needed o p oceed om 1 Φ o 2 Φ, is called phase ansi ion poin (p p). Clays ha e a su ace cha ge densi y o abou one cha ge pe nm 2 . Ca ionic su ac an s, like TTMAB , bind o he su ace and compensa e he cha ge densi y o he clay pa icles. In he case o 1 w % Saponi e 7 mM TTMAB a e necessa y [18]. Clay pa icles a e s i and do no show any undula ion o ces like su ac an bilaye s. I is concei able ha bound su ac an s a e no andomly dis ibu ed o all a ailable clay su aces and ionic cha ges, bu he su ac an s bind in a coope a i e manne . This means ha some clay pla ele s migh be comple ely co e ed while o he s a e s ill comple ely ee o su ac an s. We also examined he in e ac ion be ween a ypical anionic su ac an like Sodium-Dodecylsul a e (SDS) and he clay Saponi e. Su ace ension measu emen s o he supe na an s o SDS-Clay mix u es esul ed in he same alues as pu e SDS solu ions. The a ac i e hyd ophobic in e ac ion be ween he C 12 -chain o SDS and he clay is ob iously oo small o o e come he elec os a ic epulsion be ween bo h. The a io o bo h ene gies is de e mining, i adso p ion o he clay pa icle is happening o no . I is also well known ha non-ionic su ac an s bind o clays [19]. This adso p ion can only be explained by hyd ophobic in e ac ion be ween bo h pa icles. Fo hese expe imen s we used a ypical non-ionic su ac an like C 12 EO 7.8 . Wi h inc easing he su ac an concen a ion in clay solu ions we obse e adso p ion esul ing in clea solu ions, ollowed by p ecipi a ion and e-dissolu ion. The e o e h ee phase ansi ions poin s can be de ec ed ( ab. 1). E en he use o zwi e ionic su ac an s like he alkylaminoxides esul s in simila phase beha iou like in ig. 1. In ou s udy we used Te adecyldime hylaminoxide (TDMAO). This su ac an can no compensa e he cha ge o he clay, bu ye i binds o he 101 clay, he binding leads o p ecipi a ion and wi h excess o TDMAO leads o e-dissolu ions o he p ecipi a es. The binding o TDMAO and C 12 EO 7.8 can no occu due o elec os a ic in e ac ion. I is he e o e likely ha hese su ac an s bind due o hyd ophobic in e ac ion. This means ha he su ace o he clays has o be a leas weakly hyd ophobic. In able 1 he p p, smac, cmc and scc o di e en su ac an s a e summa ized. These esul s show unambiguously ha wi h he excep ion o anionic su ac an s all o he ypes o su ac an s bind o clays a low concen a ions wi hou o ming any p ecipi a es in he samples. Wi h inc easing su ac an adso p ion he epulsi e in e ac ion due o he ionic cha ge o he clays can be compensa ed by he a ac i e in e ac ion due o he bound su ac an molecules. I can be a gued ha he su ac an s do no bind andomly on he su aces o he clays bu o m hemi- micelles. This may be possible, bu hese hemi-micelles a e hen o med a concen a ions whe e he su ac an s do no o m micelles in he bulk phase. The s ong binding is he e o e also an indica ion ha he su ace o he clays is al eady hyd ophobic. Clays should he e o e ha e su ace ac i e p ope ies and adso b a he su ace o an aqueous phase. The eason why hey do no lowe he su ace ension has o do wi h hei la ge size and hei small numbe densi y wha esul s in a low su ace p essu e (see heo e ical pa o he pape ). 3.1.2 Clays and polyme s Wha has been shown o he binding o su ac an s o clay pa icles is also ue o amphiphilic polyme s. All wa e - soluble amphiphilic polyme s bind o clays. A high concen a ions hey sa u a e he clay su aces comple ely. Polyme s can be classi ied in o (block) copolyme s, p o eins and o he polyme s, like poly inyl alcohol. The su ace ension p o ile o clay-polyme solu ions is simila o he one shown o su ac an s ( ig. 1). O cou se polyme s a e la ge molecules wi h high molecula weigh s compa ed o su ac an s. As a consequence he equi ed mola concen a ion o polyme monolaye adso p ion (pmac) is lowe compa ed o smac. I is he e o e mo e con enien o gi e he polyme s’ concen a ions in % han in mola concen a ions. Mo eo e some polyme s ha e no cmc, while o he like Plu onics [20] o ß-casein [21] ha e. The su ace ac i i y and lipophilici y o blockcopolyme s like polaxome s, also known as Plu onics, depend e y much on he empe a u e [22-23]. The compounds o m micelles and a highe concen a ions liquid c ys alline phases. Solu ions o he compounds show a sol/gel ansi ion wi h inc easing empe a u e, which is caused by he dehyd a ion o he PO- g oups. I has been shown ha a ep esen a i e om his class o compounds, namely Plu onics F 127, adso bs s ongly on Saponi e [24]. On sa u a ion abou he same weigh o he blockcopolyme is bound o he clay. P ecipi a es o clay/blockcopolyme s we e also obse ed in he solu ions. Fu he mo e we in es iga ed he in e ac ion be ween he copolyme polyoxye hylene 17 polyoxyp opylene 4 dime hyle he (AQ 1704) and Saponi e. Su ace ension measu emen s esul ed in simila esul s as pe o med be o e indica ing ha a s ong adso p ion o AQ 1704 o 1 w % Saponi e akes place. Up o 0.1 w % AQ 1704 can adso b o 1 w % Saponi e be o e he su ace ension s a s o dec ease om he wa e alue. An in e es ing g oup o polyme s om biological o igin a e p o eins. These biopolyme s a e usually su ace ac i e and he e o e lowe he su ace ension o wa e [25]. I has ecen ly been shown ha e en nega i ely cha ged p o eins like H S a P o ein B ® (HPB) bind on nega i ely cha ged clays. So p ecipi a es we e obse ed in hese samples e en hough bo h componen s a e wa e soluble [15]. O he polyme s like poly inyl alcohol (PVA) [26], poly inylpy olidon (PVP) [27] and e en he a he hyd ophilic polyme polye hylenglycol [13] bind o clay pa icles. The adso p ion can easily be de ec ed by su ace ension measu emen s. When he polyme s ca y posi i e cha ges like in DADMAC, he complexes p ecipi a e and e- dissol e wi h excess polyme [28]. The cha ge on he clays can he e o e be e e sed by such polyme s. Tab. 2 p o ides an o e iew abou he phase ansi ion poin (p p), he polyme monolaye adso p ion concen a ion (pmac), he c i ical micella concen a ion (cmc) and he polyme -clay c i ical micella concen a ion (pcc) o di e en polyme s. 3.2 Picke ing emulsions 3.2.1 Using Amphiphile co e ed clays Picke ing emulsions p epa ed om ecombinan ly p oduced hyd ophobin (HPB) and clay (Laponi e XLG) wi h amazing p ope ies ha e ecen ly been epo ed [15]. The ob ained high in e nal (o/w) phase emulsions (65 w % oil) a e e y s able and ha e gel-like p ope ies. The s o age modulus G’ is en imes la ge han he one o he emulsion, which has been p epa ed om he hyd ophobin alone. The clay-hyd ophobin a io can be a ied om 6:4 o 2:8 wi hou unde going big changes o he gel p ope ies. The size o he oil d ople s in he emulsion depends e y much on he shea a e ha is used in he emulsi ica ion p ocess. The diame e can easily be a ied om 20 o 1 µm. The pola i y o he oil did no seem o in luence he s abili y and he elas ici y o he samples [15]. In he p e ious in es iga ion, i was shown ha p ac ically all o he clay and he p o ein can end up a he su ace o he d ople s, i he shea a e is high enough. When p o eins bind o clays, he o he side o he a ached p o ein emains su ace ac i e. I hese pa icles bind o an in e ace, he o he side o he a ached side is s ill su ace ac i e. I is he e o e no unexpec ed ha such sys ems ha e ideal p ope ies o o m s able Picke ing emulsions. Su p isingly heses sys ems ha e no been ecognized ea lie in emulsion science. Due o hei s icky cha ac e , he p o ein-clay pa icle co e ed oil d ople s can bind o each o he and o m in his way s able, homogenous emulsions wi h gel-like p ope ies. The s o age moduli a e de e mined by he elas ic p ope ies o he hin ilm. The ilms a e ex emely s able and elas ic, e en he oil and wa e can be emo ed wi hou collapse o he h ee dimensional ilm s uc u es [15]. In his in es iga ion we wan o ind ou , i his no el p o ein- clay syne gism is unique o i i is possible o eplace he 102 hyd ophobin o ei he su ac an o polyme compounds. The e o e we p epa ed high p essu e emulsions om 1:1 mix u es o amphiphile (0.5 w %) and clay (0.5 w %). The ob ained samples p epa ed a a p essu e o 300 ba a e shown in Fig. 2. All samples con ained a inal composi ion o 0.5 w % amphiphile, 0.5 w % Laponi e XLG and 50 w % o he oil polydime hylsiloxane (PDMS). The used amphiphiles o he Picke ing emulsions in ig. 2 anged om C 16 -T ime hylammoniumb omid (CTAB), Iso idecyloc ae hylenglycole he (C 13 O 8 ), Te adecyldi- me hylaminoxide (TDMAO), Plu onic F38, o H S a P o ein ® B (HPB), poly inylalcohole (PVA; Poly iol ® LL 2860), poly inylpy olidon (PVP) and polydiallyldime hyl- ammoniumchlo ide (DADMAC). In e es ingly all samples beside he sample p epa ed wi h DADMAC a e homogenous emulsions. Mo eo e he samples wi h CTAB, C 13 O 8 , TDMAO, HPB, PVA and PVP do no o m a smoo h, ho izon al meniscus, indica ing hei gel-like p ope ies. Especially he oo hpas e like pe o mance o he Picke ing emulsion p epa ed om PVA can be clea ly seen. In o de o make a deepe compa ison o hese Picke ing emulsions, ab. 3 con ains he s o age modulus G’ (τ= 0.5 Pa and = 1 Hz) and he needed shea s ess τ [Pa] o b eak he elas ic (i p esen ) beha iou o he emulsion. Tab. 3 con i ms wha was al eady seen om Fig. 2. Fascina ingly, he samples wi h CTAB, C 13 O 8 , TDMAO, PVA and PVP ha e all gel-like p ope ies and he e o e show a simila syne gism like in he case o HPB and clay. Fo CTAB only 14 mM in combina ion wi h 0.5 w % clay a e enough o ob ain gel-like p ope ies wi h al eady 50 w % oil. One could a gue ha he clay concen a ion in he wa e phase is al eady 1 w % and he e o e somehow nea o he clay gel- ansi ion. Howe e , he syne gis ic use o Plu onic F38 o DADMAC wi h clay did no esul in gel-like Picke ing emulsions, he measu ed G’ alues a e e y low. So he a gumen ha he clay could be gel-like in he wa e phase and he e o e s abilize he emulsion is no ue. In emulsion science he samples’ appea ance in dependence o s o age ime is o g ea in e es . The e o e he samples we e incuba ed a oom empe a u e o 2 mon hs (Fig. 3). Beside he samples con aining TDMAO and Plu onic F38 all o he emulsions did no change a e 2 mon h s o age a oom empe a u e. The emulsion wi h TDMAO eleased oil, while he emulsion p epa ed wi h Plu onic F38 sepa a ed in o wo phases. We conclude ha i is indeed possible o ob ain simila gel- like Picke ing emulsions wi h o he amphiphiles beside HPB. Mo eo e ou s udies show ha by he choice o he amphiphile one can easily adjus he G’ alue o he Picke ing emulsion. These esul s o e a lo o possibili ies, chances and independence o applican s. Howe e , he gene al ques ion abou he o igin o ha amphiphile-clay syne gism comes up o scien is s. In he case o he p o ein HPB, we a gued ha he oil d ople s a e co e ed wi h p o ein-clay sandwiches ac ing as s icky d ople s. These pa icles in e ac , in e pene a e and en angle wi h each o he esul ing in he o ma ion o a igid, h ee-dimensional ne wo k. This assump ion was p o ed by se e al expe imen s. Fi s o all he Picke ing emulsion aged wi h ime. The s o age moduli doubled a e one day indica ing ha a p og essed s i ening o he p o ein ne wo k ook place. Mo eo e i was possible o emo e all o he oil and wa e by eeze d ying. The whi e d ying esidue was obse ed wi h Scanning elec on mic oscopy (SEM). A ne wo k s uc u e wi h holes was obse ed. The size o he holes was in ag eemen wi h he emulsion d ople size. Finally he Picke ing emulsions con ac a e being dilu ed wi h he same amoun o wa e again. Ob iously an a ac i e o ce be ween he emulsion d ople s mus exis . I his s icky d ople concep is also ue o o he amphiphiles will be checked now. 3.2.2 Va ia ion o amphiphile concen a ion The ype o amphiphilic compounds used in his s udy di e s a lo in size, mola mass and beha iou . Consequen ly he co e age o he clays was qui e di e en . In o de o s udy he in luence o he clay co e age by amphiphile, we p epa ed samples wi h inc easing amoun o amphiphile, while he clay and oil con en we e kep he same. As he Picke ing emulsion p epa ed om he simple, ca ionic su ac an CTAB and clay had gel-like p ope ies, i is in e es ing o look close o his phenomenon. In o de o p o e, i e en o he ca ionic su ac an s a e able o p oduce s able gel-like emulsion in syne gism wi h clay, we used TTMAB in his sec ion. Fi s o all i is essen ial o know he co e age o he clay a a de ined amphiphile concen a ion. The e o e we pe o med su ace ension measu emen s o TTMAB -Laponi e XLG solu ions and compa ed hem o a pu e TTMAB solu ion (Fig. 4). The phase ansi ion poin (p p) om 1 Φ o 2 Φ o clay-TTMAB solu ions is eached wi h al eady 0.6 mM TTMAB . 3 mM TTMAB a e needed o co e he su ace o 0.5 w % Laponi e XLG wi h a monolaye (smac). The cmc o TTMAB is eached a 3.7 mM, whe eas he su ac an -clay cmc (scc) is ob ained a 15 mM TTMAB . Consequen ly 0.5 w % Laponi e XLG can bind 9 mM su ac an . In o de o in es iga e he in luence o he clay co e age a io on he emulsion p ope ies, high p essu e emulsions wi h inc easing amoun s o TTMAB ha e been ob ained. Picke ing emulsions con aining 0.5 mM (1 Φ), 1.5 mM (2 Φ), 5 mM (abo e smac), 10 mM and 45 mM TTMAB (abo e scc) a e shown in Fig. 5. All samples con ain 0.5 w % Laponi e XLG and 50 w % PDMS as an oil. Wi h inc easing concen a ion o TTMAB he olume o he emulsion laye enla ged. The ex ension o he emulsion laye can be explained wi h he dec easing concen a ion o unco e ed clay in he wa e phase. The osmo ic p essu e he e o e would be ge ing smalle , he emulsion laye can swell. P esump ion o his is a coope a i e binding o he su ac an o he clay. Mo eo e i is also possible ha an enhanced en anglemen o he oil d ople s due o he mo e co e ed clay pa icles wi h su ac an is esponsible ha he igh ening o emulsion ne wo k is ge ing lowe . Ne e heless, al eady as less as 5 mM TTMAB we e enough o ob ain a homogenous emulsion. The samples wi h 5 and 10 mM TTMAB show no ho izon al meniscus indica ing hei gel-like p ope ies. Inc easing he TTMAB concen a ion o e he scc (15 mM) leads o dec eased elas ic p ope ies as can be seen om la meniscus 103 o he sample con aining 45 mM TTMAB . In o de o look close o he heological p ope ies o he Picke ing emulsions p epa ed om di e en ly su ac an -co e ed clay pa icles we measu ed and compa ed he s o age moduli G’ (τ = 0.05 Pa and = 1 Hz) agains he used TTMAB concen a ion (Fig. 6). Adding li le amoun s o TTMAB leads o inc eased s o age moduli. A e eaching he smac o he clay pa icles, G’ is e en ge ing sligh ly highe han be o e, bu hen s a s o dec ease d ama ically. Adding mo e and mo e su ac an (abo e scc) leads inally o 100 imes smalle G’, indica ing he b eakdown o he emulsion gel-like p ope ies. As i is known om ou p e ious in es iga ion Laponi e XLG does nei he s abilize an emulsion i sel no does show any gel- ansi ion a as low concen a ions o 0.5 w % [15]. The e o e he shown esul in Fig. 6 clea ly indica es ha he clay co e age has immense in luence on he heological p ope ies o he sample. While adding small amoun o su ac an can boos he emulsi ying p ope ies o he solu ion, high amoun s dec ease hem apidly. Be o e eaching he smac, he clay pa icles became mo e hyd ophobic due o enhanced TTMAB binding. Consequen ly he clay-su ac an pa icles a e mo e likely o bind o an oil-wa e in e ace. Thei adso p ion ene gy becomes la ge han he epulsi e ene gy. Clay pa icles co e ed wi h a su ac an monolaye a e comple ely hyd ophobic and he e o e o m emulsions wi h he highes gel-like p ope ies. In Fig. 7 a heog am a τ = 0.5 Pa o he emulsion con aining 5 mM TTMAB , 0.5 w % Laponi e XLG and 50 w % PDMS is shown, indica ing i s high gel-like p ope ies. Adding mo e su ac an leads o mino gel-like p ope ies as su ac an bilaye s build up, esul ing in inc eased hyd ophilic p ope ies o he clay-su ac an pa icles again. A e eaching he scc, su ac an bilaye s a e adso bed on each side o he clay molecules, making hem posi i ely cha ged. These pa icles a e likely o bind ha dly o an oil-wa e in e ace. The e o e he emulsion p epa ed om 45 mM TTMAB is only iscous, no s icky d ople s a e o ming a ne wo k. The oil d ople s a e co e ed by TTMAB molecules, making he emulsion d ople s posi i ely cha ged. The emulsion is now s abilized by elec os a ic epulsion and no longe by emulsion d ople a ac ion as i was be o e. Clay-su ac an pa icles do no ac longe as s icky d ople s. Finally we can conclude ha by adjus ing he concen a ion o su ac an , one has an ad an ageous ool o egula e easily he heological p ope ies o he desi ed emulsion. 3. Compa ison o Picke ing emulsions om clays wi h o he Picke ing emulsions I is in e es ing o compa e he p epa a ion and p ope ies o Picke ing emulsions om his in es iga ion wi h Picke ing emulsions which ha e been p epa ed om silica Nanopa icles [29]. The g oup o Binks has used comme cially a ailable silica pa icles o he p epa a ion o Picke ing emulsions. The silica pa icles had an a e age diame e o 10 nm and a a pH = 9.5 a simila cec like clays. Aqueous solu ions o he pa icles we e anspa en and o low iscosi y. Wi h inc easing concen a ion o CTAB a p ecipi a e o med in he samples which eached a maximum a ound he cec and hen dec eased again wi h excess CTAB. The sedimen is howe e no edispe sed up o 100 mM CTAB in 2 % silica solu ions. This beha iou is he same as o he obse ed Laponi e/TTMAB in e ac ion in ou in es iga ion. The samples wi h 2 % silica pa icles showed e en a CTAB concen a ion egion in which he sys em was s ill clea and no p ecipi a e was obse ed e en hough he CTAB was adso bed on he silica pa icles. Su ace ension measu emen s showed ha he silica pa icles did no lowe he su ace ension o wa e like he clay pa icles. The samples wi h CTAB below he cec showed howe e a educed su ace ension o 68 mN/m. I is concei able ha his educed su ace ension was due o he silica pa icles which had become sligh ly hyd ophobic by he adso bed su ac an . I is unlikely ha i was due o ee CTA-ions. Emulsions we e p epa ed in samples wi h 2 % silica pa icles and inc easing CTAB concen a ion om 0.1 mM o 100 mM. All samples consis ing ou o 50 % dodecane and 50 % aqueous phase. They esul ed in a wo laye sys em, a lowe aqueous laye and an uppe o/w laye . The olume ac ion o he emulsion phase inc eased wi h he su ac an concen a ion up o he cec and dec eased again o highe su ac an concen a ion. The s abili y o he emulsions agains coalescence was highes a he CTAB concen a ion a ound he cec ha is when he silica/su ac an pa icles had no cha ge. No de ailed heological da a we e epo ed. I was men ioned howe e in he manusc ip ha he iscosi y o he emulsions passed o e a maximum wi h he CTAB concen a ion. The mos ema kable di e ence be ween he Picke ing emulsions om clay and om silica pa icles is ha he one wi h clays ha e s ong gel-like p ope ies a small co e age o he pa icles wi h su ac an while he Picke ing emulsions wi h silica pa icles beha e like iscous solu ions. In he i s si ua ion he in e ac ion be ween he d ople s is a ac i e while i is epulsi e in he second case. While i is no comple ely clea a p esen whe e his di e ence in beha iou comes om, i is concei able ha i lies in he di e ence in sizes o he wo in e ac ing pa icles. Fo he clays, he su aces a e la and ha e a size o abou 40 nm x 40 nm = 1600 nm 2 while o he silica pa icles he in e ac ing su ace o he sphe e is in he ange o 2 = 25 nm 2 . Each clay pa icle is co e ed wi h much mo e amphiphile molecules and he e o e a s ickie pa icle han he ones p epa ed wi h silica. This su ely in luences he gel-like p ope ies o emulsions based on amphiphile co e ed clay o silica pa icles. 4. Theo e ical pa - Why soluble hyd ophobic clay pa icles seem o be no su ace ac i e Hyd ophobic molecules like su ac an s, p o eins o amphiphilic polyme s a e su ace ac i e and lowe he su ace ension o wa e . These molecules o m monolaye s a he aqueous su ace and hei su ace p essu e π o he monolaye s ac s agains he su ace ension o wa e σ°. The su ace ension σ o he solu ion is hus σ ° - π. The su ace p essu e π is gi en by he equa ion 104 π = Γ . RT (1) whe e Γ is he su ace concen a ion ha is he numbe o moles pe a ea A. Fo small molecules like su ac an and polyme s he su ace concen a ion Γ is high enough when he molecules o m a monolaye so ha he su ace ension is educed. E en when he adso bed molecules a e s ill in he gaseous s a e he su ace p essu e is al eady app eciable and can be measu ed by he Langmui Blodge echnique wi h molecules ha a e no soluble in wa e . When hyd ophobic pa icles like clays a e adso bed hei su ace concen a ion Γ is so low ha he esul ing su ace p essu e is so small ha i does no a ec he su ace ension. This si ua ion mus exis wi h he clay solu ion wi h 0.5 w % o clay whe e up o 3 mM o TTMAB could be adso bed wi hou any de ec able lowe ing o he su ace ension (Fig. 4). Ob iously, all he added su ac an had been adso bed by he clay because he cec o he clay is 0.6 meq/g. The clay- samples wi h mo e han 0.6 mM TTMAB o m a p ecipi a e. Ob iously, hey ha e become so hyd ophobic by he adso p ion ha hey p ecipi a e in spi e o he ac ha he pla ele s s ill ha e an elec ic double laye and he long ange in e ac ion be ween hem is epulsi e. Wi h less hen 0.6 mM TTMAB he clay solu ions a e anspa en e en hough he su ac an is adso bed as is shown by he alue o he su ace ension (Fig. 4). We can he e o e conclude ha he hyd ophobici y in his case is no big enough o o e come he elec os a ic epulsion be ween he clays. Clays wi h 20 % adso bed su ac an o hei cec a e al eady so hyd ophobic o p ecipi a e. I is concei able ha he clays hemsel es a e al eady hyd ophobic and hei hyd ophobici y is one o he easons why su ac an s bind on clays. This si ua ion may exis only when he clays a e dispe sed in wa e and mos o he coun e -ions a e dissocia ed om he clay su ace. The si ua ion can be di e en wi h d y clays when he coun e -ions a e bound o he su ace. I is hus concei able ha dissol ed clays wi h adso bed su ac an s s ongly bind o oil d ople s in emulsions while naked clays can no well adso b on oil d ople s because he coun e -ions in his case a e in be ween he oil and he clays (Fig. 8). 5. Conclusion I was shown ha almos all s udied amphiphilic compounds bind on nega i ely cha ged clay pla ele s. Nega i ely cha ged su ac an s like SDS do no bind. Fa om he sa u a ion capaci y, he clay-amphiphile pa icles a e soluble. A ound sa u a ion he pa icles p ecipi a e. S able Picke ing emulsions can be p epa ed om samples wi h 50 % wa e and 50 % oil wi h as li le as 0.5 % clay and low amoun s o amphiphile. The Picke ing emulsions a e o he o/w- ype and ha e gel-like p ope ies. The shea modulus o hese phases can be a ied be ween a ew Pascal and se e al housand Pascal o small changes in he composi ion on he clay su ace. I is no mally assumed ha clays ha e hyd ophilic su aces because clay pa icles do no lowe he su ace ension o wa e . Amphiphilic molecules like non-ionic su ac an s o su ace ac i e wa e soluble polyme s bind o clay su aces. The esul ing amphiphile co e ed clay pa icles again do no lowe he su ace ension o wa e e en hough hese pa icles should now ha e hyd ophobic p ope ies. To explain hei con lic ing e idence, i is concluded ha he clay pa icles and he su ace modi ied clay pa icles a e eally hyd ophobic bu do no lowe he su ace ension o wa e because hei su ace p essu e is no big enough o educe he su ace ension o wa e . The su ace p essu e is gi en by he numbe densi y o he adso bed pa icles and in he case o he clays hei numbe densi y is se e al o de s o magni ude lowe han he numbe densi y o su ac an s. The su ace ension is he e o e p ac ically no a ec ed. I is o his hyd ophobici y o he modi ied clay pa icles ha hey o m e y s able Picke ing emulsions wi h oil and he emulsions ha e gel-like p ope ies. The clay pa icles a e c oss-linking he oil d ople s. 6. 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[12] He e a N, Le o e J-M, Bou gea -Lami E, Langmui , 2004; 20; 1564-1571. [13] Zhao X, U ano K, Ogasawa a S, Colloid & Polyme Science, 1989; 267; 899-906. [14] Hu X, Wang T, Tong Z, Langmui , 2010; 26; 4233- 4238. [15] Rege M, Sekine T, Ho mann H, So Ma e , 2011; submi ed [16] Wohlleben W, Subkowski T, Baus U, Eu opean Biophysics Jou nal, 2009; 39; 457-468. [17] Binks B P, Rod igues J A, Angew. Chem. In . Ed., 2007; 46; 5389-5392. [18] Yamaguchi Y, Ho mann H, Colloids and Su aces A, 1997; 121; 67-80. [19] Alexand idis P, A hanassiou V, Ha on A, Langmui , 1994; 10; 2604-2612. 105 112 8 Danksagung Ich möch e mich he zlich bei He n P o . D . Heinz Ho mann ü die seh in e essan e Themens ellung sowie die in ensi e Be euung wäh end meine Disse a ion bedanken. Seine e sie e, wissenscha liche Au assungsgabe sowie sein une schöp liche Ideen eich um suchen Ih esgleichen. De japanischen Kosme ik i ma Shiseido danke Ich ü die kon inuie liche Finanzie ung wäh end de expe imen ellen A bei en zu meine Disse a ion. Die Besuche und Diskussionen mi D . Kei Wa anabe und Tomoko Sekine wa en übe aus nü zlich ü den Fo gang meine A bei und de Ve ö en lichungen. Ge ne denke Ich an den gemeinsamen Labo all ag mi den Japane n Takashi, Keisuke und Yoko zu ück. Na ü lich möch e Ich mich bei meinem Kollegen und langjäh igen F eund Lukas Wol bedanken. Obwohl e mi seine P omo ion seh ge o de wa , ha e sich imme aus eichend Zei ü meine F agen genommen. Danke Di , ü die Leich igkei an manch zähen Tagen. Ein Dank gil auch meinen ande en Labo kollegen: Die e , Elham, Kalle und Rami. Zudem möch e Ich mich bei He n P o . D . Ge ha d Pla z ü seine ausdaue nde Be ei scha mich au dem Feld de physikalischen Chemie kon inuie lich o wä s zu b ingen, ech he zlich bedanken. De BASF, o allem abe D . Ul Baus, D . Ma in Ka os und D . Be nd Reck danke Ich neben de Be ei s ellung de Hyd ophobine auch ü die in o ma i en und diskussions eichen Besuche in Ludwigsha en. Da Ich mich expe imen ell o an de Uni e si ä Bay eu h au hiel , gil mein Dank neben ielen ande en: D . Bea e Fö s e , D . Ma kus D echsle und Ma ina Heide sowie den Leh s ühlen und Mi a bei e n on P o . Fe y, P o . Fö s e , P o . Rösch und P o . Scheibel. G öß e Dank gil meine Familie, meinem leißigen Va e Albe , meine he zensgu en Mu e Angela sowie meinem g andiosen B ude Jü gen ü die imme wäh ende Hil e. Ein he zliches „Ve gel ’s Go “ auch an die gesam e Familie Schlich . Meinen bes en F eunden Michl, Maksi, Olli, Ca o und Dominik danke Ich ü Ih e jah elange Un e s ü zung. Danke Di , eue Mo i z. Wo S ü me des Lebens wal en, da b auch es Menschen, die wie Felsen s ehen. Du, liebs e Susanne, gibs mi den Hal , nach dem Ich mich imme gesehn habe. Deine Bescheidenhei , deine Liebe und dein geduldiges Ve s ändnis sind ein Spiegelbild Deine kla en Seele. Ich liebe Dich, meine Lilie. 113 9 E klä ung Hie mi e klä e ich, diese A bei selbs s ändig e ass und keine ande en als die angegebenen Quellen und Hil smi el benu z zu haben. Fe ne e klä e ich, dass ich nich ande wei ig mi ode ohne E olg e such habe, diese Disse a ion einzu eichen. Ich habe keine gleicha ige Dok o p ü ung an eine ande en Hochschule endgül ig nich bes anden. Immen eu h, den 5. Dezembe 2011