1
E ec o cinnamaldehyde on in e acial heological p ope ies o
p o eins adso bed a O/W in e aces
Manuel Felixa,*, Jack Yangb, An onio Gue e oa and Leona d M. C. Sagisb
a Depa amen o de Ingenie ía Química, Escuela Poli écnica Supe io , Uni e sidad de Se illa, 41011
Se illa, Spain.
b Labo a o y o Physics and Physical Chemis y o Foods, Wageningen Uni e si y, Bo nse Weilanden 9,
6708WG Wageningen, The Ne he lands.
______________________
*M. FELIX
Depa amen o de Ingenie ía Química,
Uni e sidad de Se illa, Facul ad de Química,
41012 Se illa (Spain)
E-mail: m [email p o ec ed]
Phone: +34 954557179; ax: +34 954556447.
2
Abs ac 1
The dynamics o he e ogeneous ood p oduc s such as emulsions o oams can be a ec ed 2
signi ican ly by he in e acial p ope ies o hei in e aces. P o eins a e widely used o inc ease 3
he s abili y o hese ood p oduc s. This wo k compa es he in e acial p ope ies o a model 4
p o ein (whey p o ein isola e, WPI) and silkwo m pupae (SLW) adso bed a he O/W in e ace. 5
A na u al aldehyde (cinnamaldehyde, CNM) was used o bo h p o ein sys ems in o de o 6
p omo e p o ein-p o ein in e ac ions. In e acial p ope ies we e cha ac e ised du ing p o ein 7
adso p ion and a e eaching a quasi-equilib ium s a e by means o oscilla o y and s ep 8
dila a ional, and oscilla o y in e acial shea measu emen s. The esul s ob ained om dila a ional 9
and in e acial shea es s showed ha he use o CNM esul ed in he de elopmen o s onge 10
in e aces, wi h highe alues o he dila a ional and su ace shea s o age moduli, and a lowe 11
loss angen . S ep-dila a ion es s indica ed ha he addi ion o CNM also esul ed in mo e 12
homogeneous in e aces. Ou esul s show ha CNM addi ion can enhance he su ace p ope ies 13
o SLW, o a le el which is close o he p ope ies o un-modi ied WPI s abilized in e aces. 14
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Keywo ds: Dila a ional; In e acial shea ; LAOD; Lissajous 26
3
1. In oduc ion 27
Many ood p oduc s consis o a kine ically s able combina ion o wo phases (A/W o O/W). 28
These p oduc s equi e he use o su ace ac i e agen s o a oid hei des abiliza ion (which is 29
d i en by he modynamic e en s) (Damoda an, Pa kin, & Fennema, 2007). P o eins a e widely 30
used o achie e kine ic s abili y o ood p oduc s. The ood indus y p e e s using p o eins a he 31
han low molecula weigh su ac an s (LMWS), since p o eins ha e be e consume accep ance, 32
ha e nu i ional added- alue, and a e able o modi y heological p ope ies o he con inuous 33
phase since hey can in e ac wi h each o he (Tad os, 2013). When p o eins adso b a he 34
in e ace, hey educe he in e acial ension and hey a e able o o m complex luid- luid 35
in e aces, dec easing hei la e al mobili y (Fulle & Ve man , 2012). The adso p ion o p o eins 36
a O/W in e aces o en esul s in hei un olding and consequen ly, hei dena u a ion. A e ha , 37
he numbe o exposed hyd ophobic esidues, he abili y o de elop p o ein-p o ein in e ac ions, 38
as well as he lexibili y o he p o ein s uc u e inc ease (Dickinson, 1989; Pa ino e al., 2007). 39
Chemical modi ica ion has been used o ailo he hyd ophobici y o ne su ace cha ge o 40
p o eins. The in-si u c osslinking o p o eins a O/W in e ace has been ca ied ou , mainly by he 41
use o enzymes (Fische & Windhab, 2011; Romoscanu & Mezzenga, 2005), o by using 42
aldehydes, which a e no ood-g ade (Ge a d, 2002). S udies using na u al aldehydes (i.e. 43
cinnamaldehyde, CNM) o p omo ing p o ein-p o ein in e ac ions a O/W in e aces a e s ill 44
sca ce. 45
Typically, ood emulsions ha e been s abilized mos commonly by he use o milk o egg 46
p o eins. Whey p o eins (WPI) om milk exhibi excellen unc ional p ope ies (i.e. high su ace 47
ac i i y) since hei globula p o eins ha e a s ong endency o ge adso bed a O/W in e aces. 48
A e p o ein adso p ion, WPI p o eins a e un olded and hei laye s a e cha ac e ised by a slow 49
eo ganiza ion s ep which in ol es he de elopmen o s ong p o ein-p o ein in e ac ions. 50
E en ually, a iscoelas ic ilm is o med, in which he p o eins a e densely packed. Consequen ly, 51
WPI p o eins p o ide emulsions wi h excellen s abili y agains physicochemical des abiliza ion 52
phenomena (Dickinson, 2003). The e is howe e an inc easing demand om consume s o he 53
4
use o non-animal p o ein sou ces. In his sense, insec p o eins ha e gained global a en ion since 54
hei use may educe he en i onmen al oo p in o p oduc s. P o ein ex ac s om insec s a e 55
soluble and hey exhibi unc ional p ope ies like wa e holding capaci y, oaming and 56
emulsi ying p ope ies (Zielińska, Ka aś, & Ba aniak, 2018). P o eins om silkwo m pupae 57
(SLW) ha e also shown nu i ional and echnological p ope ies (Kim, Se yab a a, Lee, Jones, & 58
Kim, 2016). 59
P o eins adso bed a O/W in e aces can o m in e acial laye s which may exhibi iscoelas ic 60
o e en elas ic beha iou . The cha ac e is ics o hese ilms a e key o unde s anding he s abili y 61
o p oduc s s abilized by p o eins, and in e acial heology is, he e o e, an essen ial ool o he 62
cha ac e iza ion o he sui abili y o p o eins as a s abilize o emulsions. In e acial heology 63
can be pe o med in dila a ional o in e acial shea mode. Dila a ional heological measu emen s 64
a e mos commonly pe o med using Langmui oughs o d ople ensiome e s. D ople 65
ensiome e s c ea e a d ople a he ip o a sy inge and use he Laplace equa ion o calcula e he 66
in e acial ension, ei he om he d ople p o ile, o he p essu e in he d ople . When he a ea o 67
he d ople is subjec ed o a sinusoidal change, he elas ic (o s o age) and he iscous (o loss) 68
dila a ional moduli can be de e mined om he i s ha monic o he Fou ie ans o m o he 69
oscilla ing in e acial ension signal (Bagley & To ik, 1983). 70
In e acial shea heology has also been p oposed as a ool o he cha ac e isa ion o complex 71
in e aces since i can p o ide in o ma ion abou in e - and in a-molecula p o ein-p o ein 72
in e ac ions a he O/W in e ace (K ägel, De ka ch, & Mille , 2008). Con a y o dila a ional 73
de o ma ions, in in e acial shea measu emen s, he p o ein concen a ion in he adso bed ilm 74
does no change du ing de o ma ion. They also gi e di ec access o he in-plane de ia o ic 75
s esses and a e no a ec ed by he bending igidi y o he in e acial ilm. Al hough p e ious 76
s udies deal wi h he use o CNM o inc ease he s abili y o WPI-based nanoemulsions (Chen e 77
al., 2018; Chen, Wu, McClemen s, Li, & Li, 2017) and chi osan-based nanoemulsions (Tian, Lei, 78
Zhang, & Li, 2016), hese au ho s did no cha ac e ise he changes o he in e aces when CNM 79
is p esen in he oil phase. They only de e mined he in e acial ension and analysed images o 80
he pendan d op a e comp ession. 81
5
The aim o his wo k was o es ablish he in e acial e ec s o he na u al aldehyde CNM, on 82
he in e acial heological p ope ies o O/W in e aces s abilized by WPI and SLW, and in 83
pa icula , o es ablish i his aldehyde can p omo e p o ein-p o ein in e ac ions o such an ex en 84
ha he alues o he su ace heological p ope ies a e inc eased. To assess hese e ec s, 85
oscilla o y dila a ional and in e acial shea measu emen s we e ca ied ou . In addi ion, he 86
esponse o he in e aces was p obed using dila a ional s ep de o ma ion es s. 87
2. Ma e ial and me hods 88
2.1. Ma e ials 89
BiPRO® was he whey p o ein isola e used o all measu emen s (pu i y 98%). I was supplied 90
by AGROPUR (Longueuil, Canada). Silkwo m p o ein concen a e (SLW, 50.5 ± 0.3 w .% 91
p o eins) was supplied by FeedS imulan s ( he Ne he lands), his p o ein concen a e was de a ed 92
be o e use, emo ing o he su ace ac i e agen s apa o p o ein (e.g. phospholipids) . Medium 93
chain iglyce ides (MCT) oil was used as non-pola phase. I was supplied by IOI Oleochemical 94
(F ance), con aining C8 and C10 a y acids (≥ 98%) and C6 a y acid (≤ 2%), being ee o 95
su ace ac i e con aminan s. Chemical eagen s (i.e. HCl, NaOH, NaH2PO4, cinnamaldehyde) 96
we e pu chased om Sigma–Ald ich company (S . Louis, USA). The solu ions we e p epa ed 97
using Milli-Q g ade wa e . 98
2.2. Me hods 99
2.2.1. P epa a ion o p o ein solu ions 100
Taking in o accoun p o ein con en and solubili y, WPI and SLW solu ions we e p epa ed a 101
1 w .% soluble p o ein (0.1 mg/mL) in 50 mM phospha e bu e (pH 7.0). Glasswa e was ca e ully 102
cleaned o a oid he p esence o su ace-ac i e agen s by insing i se e al imes wi h deionized 103
and Mili-Q wa e . The su ace ension o he las wash wa e was de e mined using a pendan 104
d ople ensiome e , ob aining a alue be ween 69 and 72 mN/m. P o ein dispe sions we e gen ly 105
s i ed in new plas ic con aine s a 500 pm o e 30 min a oom empe a u e, and subsequen ly 106
hey we e cen i uged o 15 min a 15,000 g. Pelle s we e disca ded and supe na an s (1 w .% 107

6
p o ein solu ions) we e s o ed a 4 ºC o e nigh . P o ein solu ions we e always used he day a e 108
p epa a ion. 109
2.2.2 D ople ensiome y measu emen s 110
D ople ensiome y measu emen s we e ca ied ou using a T acke au oma ic d ople 111
ensiome e om Teclis scien i ic (Ci ieux d'Aze gue, F ance). An axisymme ic d ople wi h a 112
olume o 8 μL and a su ace a ea o 18 mm2 was c ea ed a he ip o a e ical needle, which 113
was connec ed o a mo o ized sy inge. The d ople p o ile was digi ized e e y 0.01 s using a 114
digi al came a connec ed o a compu e . D ople p o iles we e p ocessed using he Laplace 115
equa ion (Cas ellani, Al-Assa , Axelos, Phillips, & An on, 2010). All expe imen s we e ca ied 116
ou using a low-abso bance glass cu e e (8 ml). D ople expe imen s we e ca ied ou in absence 117
o CNM and adding 2 w .% CNM o he oil phase. These expe imen s we e pe o med a 20.0 ± 118
0.1 °C. 119
Dila a ional p ope ies in he p o ein adso p ion phase 120
In e acial ension was de e mined o e a pe iod o 10,800 s o moni o he p o ein adso p ion 121
kine ics. Oscilla o y dila a ional expe imen s we e ca ied ou du ing he p o ein adso p ion 122
phase, e e y 500 s. The oscilla ions ( i e cycles) we e ca ied ou a 5 % ampli ude and a a 123
equency o 0.1 Hz, which was es ed o be wi hin he linea iscoelas ic egime ( esul s no 124
shown). 125
Dila a ional p ope ies in he quasi-equilib ium phase 126
Oscilla o y dila a ional expe imen s we e ca ied ou o ob ain he mechanical spec a o he 127
p o ein-s abilized O/W in e aces a e eaching a quasi-equilib ium s a e (10,800 s). F equency 128
sweep es s we e pe o med om 0.075 Hz o 0.1 Hz a 5% ampli ude, o ob ain he equency 129
dependence o he in e acial iscoelas ic moduli (E’i and E’’i). The dependence o he E’i on 130
equency was analysed wi h he ollowing powe law equa ion: 131
𝐸𝐸𝑖𝑖
′=𝑘𝑘·𝜔𝜔𝑛𝑛′ (1) 132
whe e 𝜔𝜔 is he equency and 𝑛𝑛′ is he slope in a double loga i hmic ep esen a ion. 133
7
S ep dila a ion es s we e also pe o med a e he p o ein adso p ion phase. In hese es s he 134
in e aces we e subjec ed o a s ep expansion o 10 o 20 % o he a ea, and subsequen ly he 135
elaxa ion o he in e acial ension was moni o ed o 1,000 s. A e his leng h o ime, he 136
in e acial ension alues we e no longe changing signi ican ly (p < 0.05) and he a ea was 137
comp essed by 10 o 20 % (comp ession s ep). Again, he e olu ion o he in e acial ension was 138
moni o ed o 2,000 s. Resul s we e i ed using Ma lab R17a (Ma hwo ks, USA) o he ollowing 139
exponen ial equa ion, which combines a Kohl ausch-Williams-Wa s s e ched exponen ial e m 140
wi h a egula exponen ial e m (Sagis e al., 2019): 141
𝛾𝛾(𝑡𝑡)=𝑎𝑎𝑎𝑎−(𝑡𝑡/𝜏𝜏1)𝛽𝛽+𝑏𝑏𝑎𝑎−𝑡𝑡/𝜏𝜏2+𝑐𝑐 (2) 142
he e γ is he in e acial ension, 𝜏𝜏1 is he ini ial elaxa ion ime, β is he s e ch exponen , 𝜏𝜏2 is a 143
cha ac e is ic ime o ageing p ocesses, which a e also p esen when he in e acial a ea is no 144
pe u bed by a s ep dila a ion. The pa ame e s a, b, and c a e cons an s. 145
2.2.3. Shea measu emen s 146
In e acial shea heology was ca ied ou using an AR-G2 heome e om TA Ins umen s (New 147
Cas le, USA), wi h a double-wall- ing geome y (DWR). The p o ein solu ions we e pou ed in a 148
double wall cup and he ing was posi ioned a he ai /wa e in e ace. Immedia ely a e wa ds, 149
MCT oil was ca e ully added on op o he aqueous phase o a oid any dis u bance o he 150
in e ace. All shea expe imen s we e ca ied ou using oil phases wi hou CNM and oil con aining 151
2 w .% CNM. All hese expe imen s we e ca ied ou a 20.0 ± 0.1 °C. 152
Shea p ope ies du ing he adso p ion phase 153
In e acial iscoelas ic moduli we e ob ained du ing he p o ein adso p ion phase (10,800 s) 154
wi hin he linea iscoelas ic egime (LVR). These measu emen s we e ca ied ou in s ess-155
con olled mode, wi h an ampli ude o 1·10-6 Pa·m o he oscilla ion s ess and a equency o 156
0.01 ad/s (which was con i med o be in he LVR by pe o ming s ain sweep es s). 157
Shea p ope ies in he quasi-equilib ium s a e 158
Small ampli ude oscilla o y measu emen s (SAOS) we e ca ied ou a e eaching he quasi-159
equilib ium s a e (10,800 s) o ob ain he mechanical spec a o he O/W in e aces. F equency 160
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sweep es s we e pe o med om 0.075 Hz o 0.1 Hz a 1·10-6 Pa·m s ess ampli ude (which was 161
con i med o be in he LVR by pe o ming s ain sweep es s), o ob ain he in e acial iscoelas ic 162
shea moduli (G’i and G’’i). The dependence o he G’i on equency was analysed wi h he 163
ollowing powe -law equa ion: 164
𝐺𝐺𝑖𝑖
′=𝑘𝑘·𝜔𝜔𝑛𝑛′ (3) 165
The Boussinesq Numbe (Bo) was calcula ed o assess he ela i e con ibu ions o he adjoining 166
bulk phases and he in e ace o he o al measu ed s ess (Vandeb il, F anck, Fulle , Moldenae s, 167
& Ve man , 2010). This numbe is de ined as 168
Bo =𝜂𝜂𝑖𝑖
∗
(𝜂𝜂𝑂𝑂+𝜂𝜂𝑊𝑊)∙ 𝑅𝑅 (6) 169
whe e 𝜂𝜂𝑖𝑖
∗ is he in e acial complex iscosi y (uni s Pa·s·m), and 𝜂𝜂𝑂𝑂 and 𝜂𝜂𝑊𝑊 a e he iscosi y o 170
he bulk phases (oil and wa e ) and 𝑅𝑅 is he cha ac e is ic expe imen al leng h scale (0.7 mm) o 171
he DWR geome y. The lowes obse ed alue o Bo was 27.2, and since Bo >> 1, in e acial 172
s esses domina ed he bulk esponse in all shea expe imen s (Vandeb il e al., 2010). 173
2.3. S a is ical analysis 174
A leas h ee eplica es we e ca ied ou o each measu emen . S a is ical analyses we e 175
pe o med by analysis o a iance (ANOVA) using he 95 % con idence limi o he mean. SPSS 176
22 s a is ical so wa e o Windows (Chicago, USA) was used o his analysis. S anda d 177
de ia ions we e calcula ed o some selec ed pa ame e s. 178
3. Resul s and discussion 179
3.1 D ople measu emen s 180
Dila a ional p ope ies du ing he p o ein adso p ion phase 181
Figu e 1 shows he alues ob ained o he ansien in e acial ension o he O/W in e ace 182
du ing he p o ein adso p ion phase ( < 10,800 s), wi h and wi hou cinnamaldehyde (CNM), o 183
WPI (Fig. 1A) and SLW (Fig. 1B). The kine ics o p o ein adso p ion is cha ac e ised by an ini ial 184
as dec ease in in e acial ension alues. This as dec ease was ollowed by a much slowe 185
educ ion in he in e acial ension, acco ding o p e ious s udies hese esul s can be ela ed o 186
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di e en s ages o p o ein adso p ion ( om p o ein di usion o p o ein pene a ion, un olding and 187
ea angemen ) (Manuel Felix, Rome o, & Gue e o, 2017; Pizones Ruiz-Henes osa, Ca e a 188
Sanchez, Ped oche, Millan, & Rod iguez Pa ino, 2009). These esul s illus a e ha o WPI 189
s abilized in e aces, CNM has only a ma ginal e ec on he ime-e olu ion o he in e acial 190
ension. Ini ially, he in e acial ension dec eases a a sligh ly slowe a e, and a he end o he 191
adso p ion s age, he in e acial ension is lowe when CNM is added by only abou 1 mN/m. 192
Fo SLW he di e ence is mo e subs an ial. The addi ion o CNM o he oil phase esul s in a 193
signi ican dec ease in he in e acial ension a he end o he adso p ion s age. Se e al s udies 194
indica e ha cinnamaldehyde exhibi s he abili y o in e ac wi h p o eins, causing p o ein 195
c osslinking (Balague , Gomez-Es aca, Ga a a, & He nandez-Muñoz, 2011; Sh age , 196
S ickholm, & Macey, 1969). This leads o he o ma ion o la ge p o ein species and addi ional 197
ea angemen s in he O/W in e ace (Balague e al., 2011). This could ha e esul ed in a mo e 198
densely packed s uc u e, wi h a lowe in e acial ension (Dickinson, 1999). Howe e , in WPI 199
s abilized in e aces he p o ein is al eady qui e seg ega ed and agg ega ed (Sagis e al., 2019) and 200
addi ional c oss-linking does no lead o mo e adso p ion, which would explain he insigni ican 201
d op in in e acial ension. 202
The e olu ion o dila a ional moduli o he O/W in e aces (𝐸𝐸𝑖𝑖
′ and 𝐸𝐸𝑖𝑖
′′, espec i ely) ob ained 203
om oscilla o y measu emen s du ing he p o ein adso p ion phase ( < 10,800 s), wi h and 204
wi hou CNM, can be obse ed in Figu e 2 o WPI (Fig. 2A) and SLW (Fig. 2B). 205
The dila a ional p ope ies show an inc ease in he elas ic modulus as a unc ion o ime, 206
whe eas he loss modulus unde goes a small dec ease. This endency is mo e ma ked a he 207
beginning o he p o ein adso p ion phase and i becomes mo e g adual a e a while. This 208
esponse has been p e iously ob ained o o he p o eins such as c ay ish and soy p o ein 209
concen a e, and i was ela ed o he de elopmen o a p o ein in e acial ilm (Bellesi, Pizones 210
Ruiz-Henes osa, & Piloso , 2014; Felix e al., 2017). Rega dless o he p o ein adso bed a he 211
O/W in e ace, he p esence o CNM induces a mo e p onounced inc ease o 𝐸𝐸𝑖𝑖
′, whe eas 𝐸𝐸𝑖𝑖
′′ 212
emains nea ly unal e ed. This esul suppo s he hypo hesis ha CNM is ac ing as a p omo e o 213
p o ein-p o ein in e ac ions, inc easing he elas ic beha iou o he ilm. On he o he hand, his 214
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in e acial shea heology beha iou o chickpea p o ein-adso bed complex oil/wa e 375
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432
433
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Figu e Cap ions 434
Figu e 1: in e acial ension o e p o ein adso p ion (10,800 s) a O/W in e ace wi h 435
and wi hou CNM o WPI (A) and SLW (B) p o ein sys ems. 436
Figu e 2: in e acial elas ic and iscous moduli (E’i and E’’i, espec i ely) ob ained om 437
dila a ional measu emen s o e p o ein adso p ion (10,800 s) a O/W in e ace wi h 438
and wi hou CNM o WPI (A) and SLW (B) p o ein sys ems. 439
Figu e 3: in e acial dila a ional moduli as a unc ion o equency ob ained om 440
measu emen s a e 10,800 s adso p ion a O/W in e ace wi h and wi hou CNM o 441
WPI (A) and SLW (B). 442
Figu e 4: in e acial ension o d ople s abilized by 1 % WPI a e eaching he quasi 443
equilib ium s a e (10,800 s) and being subjec ed a + 10 % expansion and – 20 % 444
comp ession in he a ea o e 1,000 s. 445
Figu e 5: elas ic and iscous moduli ( 𝐺𝐺′𝑖𝑖 and 𝐺𝐺′′𝑖𝑖, espec i ely) ob ained om 446
dila a ional measu emen s o e p o ein adso p ion (10,800 s) a O/W in e ace wi h 447
and wi hou CNM o WPI (A) and SLW (B) p o ein sys ems. 448
Figu e 6: mechanical spec a ob ained om in e acial shea measu emen s a e p o ein 449
adso p ion (10,800 s) a O/W in e ace wi h and wi hou CNM o WPI (A) and SLW 450
(B) p o ein sys ems. 451
452
20
Table 1 453
Dila a ional
Shea
Sys em
n’
an δi,0.05
n’
an δi,0.05
WPI
0.15 ± 0.01a
0.13 ± 0.01a
0.94 ± 0.03a
0.28 ± 0.01a
WPI-CNM
0.10 ± 0.01b
0.10 ± 0.01b
0.52 ± 0.05b
0.27 ± 0.01a
SLW
0.24 ± 0.02c
0.17 ± 0.02c
0.29 ± 0.02c
2.59 ± 0.11b
SLW-CNM
0.19 ± 0.01d
0.20 ± 0.01d
0.17 ± 0.01d
0.58 ± 0.05c
454
Table 1: pa ame e s (n’ and an δi) om mechanical spec a ob ained by means o 455
dila a ional and shea measu emen s wi h and wi hou CNM o WPI and SLW p o ein 456
sys ems adso bed a O/W in e ace. Di e en le e s wi hin a column indica e 457
signi ican di e ences (p < 0.05). Squa e co ela ion coe icien s (R2) ob ained om 458
linea eg ession o n’ pa ame e we e in all cases ≥ 0.98 459
460
461
21
Figu e 1 462
463
464
465

22
Figu e 2 466
467
468
469
23
Figu e 3 470
471
472
473
474
24
Figu e 4 475
476
477
478
479
25
Figu e 5 480
481