P oduc ion o wa e soluble que ce in o mula ions by p essu ized e hyl ace a e-in-wa e
1
emulsion echnique using na u al o igin su ac an s
2
V.S.S. Gonçal esa, b, c, S. Rod íguez-Rojoc, *, E. De Paz c, C. Ma o c, A. Ma ín c, M.J. Coce o c
3
a Ins i u o de Tecnologia Química e Biologica An onio Xa ie , Uni e sidade No a de Lisboa, A .
4
da República, 2780-157 Oei as, Po ugal
5
b iBET, Ins i u o de Biologia Expe imen al e Tecnologica, Apa ado 12, 2780-901 Oei as,
6
Po ugal
7
c Escuela de Ingenie ías Indus iales, Uni e sidad de Valladolid, C/D . Me gelina s/n, 47011
8
Valladolid, Spain
9
Abs ac
10
Que ce in is a s ong an ioxidan la onoid wi h se e al bioac i e p ope ies such as an i-
11
in lamma o y and an ica cinogenic ac i i ies, becoming an in e es ing compound o be
12
inco po a ed in o pha maceu ical, cosme ic o ood p oduc s. Howe e , hese applica ions a e
13
limi ed by he low bioa ailabili y o his la onoid. Que ce in is poo ly soluble in aqueous
14
media, such as gas oin es inal luids, being also deg aded by gu lo a. Thus, i is necessa y he
15
de elopmen o que ce in’s o mula ions capable o imp o ing i s wa e solubili y esul ing in
16
inc eased bioa ailabili y and hus highe biological ac i i y o his compound.
17
The aim o he p esen wo k was he o mula ion o que ce in using h ee dis inc na u al
18
o igin su ac an s, namely OSA-s a ch, Leci hin and β-glucan, by p ecipi a ion om a
19
p essu ized e hyl ace a e-in-wa e emulsion. Fo mula ions o que ce in wi h encapsula ion
20
e iciencies up o nea 76% and a micella pa icle size in he ange o nanome e s we e
21
ob ained using leci hin. An imp o ed an ioxidan ac i i y (3- old highe pe uni mass o
22
que ce in) was also obse ed in hese o mula ions, demons a ing ha leci hin is a good
23
emulsi ie o he encapsula ion o que ce in. Fu he mo e, he addi ion o glyce ol as co-
24
sol en inc eased he colloidal s abili y o he suspension and he encapsula ion e iciency
25
o he la onoid.
26
27
Keywo ds: Que ce in, OSA-s a ch, Leci hin, β-glucan, Encapsula ion, High P essu e Emulsion
28
Technique, Nanosuspension
29
30
1. In oduc ion
31
Que ce in (3,3',4',5,7-Pen ahyd oxy la one), one o he mos ep esen a i e membe o he
32
la onoid amily wi h high an ioxidan ac i i y, is commonly ound in se e al ui s and
33
ege ables like onions, apples, g apes o s awbe ies, as well as in ed wine o g een/black ea
34
[1,2]. This compound has a ac ed he in e es o he pha maceu ical and nu aceu ical
35
indus ies due o i s bioac i e p ope ies, such as an i-in lamma o y, an i-p oli e a i e and
36
neu op o ec i e e ec s [1,3]. In o de o achie e que ce in plasma’s concen a ion abo e 10
37
mM equi ed o ob aining pha macological ac i i y, he inges ion o que ce in-en iched oods
38
o supplemen s could no be enough due o he low bioa ailabili y o his la onoid [4]. The
39
low wa e solubili y (2ppm a 25ºC o 60 ppm a 100 °C) allied wi h gas oin es inal
40
deg ada ion limi s que ce in’s biological e ec s in i o [2,5]. The e a e wo main app oaches o
41
inc ease he bioa ailabili y o his compound, whe he by chemical modi ica ion o by he
42
de elopmen o colloidal que ce in deli e y sys ems [6].
43
Fo mula ions o que ce in using di e en me hods and dis inc ca ie ma e ials ha e been
44
de eloped by se e al au ho s. Rega ding polyme s, Kuma i and co-au ho s achie ed a
45
con olled elease o que ce in by i s encapsula ion in o poly-D,L-lac ide (PLA) nanopa icles
46
h ough sol en e apo a ion echnique [7]. Wu e al. ha e p oduced que ce in-loaded
47
nanopa icles by a nanop ecipi a ion me hod using Eud agi ® E and poly inyl alcohol (PVA) as
48
ca ie s, ob aining a que ce in’s elease 74- old highe in compa ison wi h he pu e compound
49
[8]. Que ce in was also encapsula ed in Plu onic F127 h ough supe c i ical an isol en me hod
50
by F aile and co-au ho s, enabling an imp o ed dissolu ion beha iou o he compound in
51
simula ed physiological luids [9]. Cyclodex ins (CD), such as α-CD, β-CD o HP-β-CD, ha e also
52
been used o he encapsula ion o que ce in using he eeze-d ying o co-e apo a ion
53
me hod [10,11]. Ba as and co-au ho s used lipids o encapsula e que ce in, being able o
54
inc ease i s appa en aqueous solubili y by a ac o o 100 [6]. Besides solid lipid nanopa icles,
55
nanos uc u ed lipid ca ie s and lipid nanoemulsions we e p oduced using a high p essu e
56
homogenize o he encapsula ion o que ce in by Adi ya e al., achie ing encapsula ion
57
e iciencies abo e 90% [12]. Que ce in-loaded liposomes ha e also been p oduced and a e
58
epo ed in he li e a u e [1,13]. Ino ganic ma e ials can also be an op ion as ca ie s o he
59
encapsula ion o que ce in and, ecen ly, que ce in-loaded silica mic osphe es we e de eloped
60
by Kim and co-au ho s using polyol-in-oil-in-wa e (P/O/W) emulsion and sol-gel me hods o
61
imp o e he la onoid’s s abili y as well as i s p ope ies [14].
62
In 2012, de Paz and co-au ho s [15] de eloped a no el me hod o he encapsula ion o
63
hyd ophobic compounds, based on he p oduc ion and p ocessing o e hyl ace a e-wa e
64
emulsions a high p essu e and empe a u e. The au ho s achie ed s able aqueous
65
suspensions o β-ca o ene wi h micella pa icle sizes down o 400 nm and encapsula ion
66
e iciencies up o 80%. Mo eo e , he ype o emulsi ie used had been shown o a ec he
67
inal p ope ies o he suspension [16]. This p ocess is an a ac i e al e na i e o he
68
con en ional emulsion e apo a ion p ocess, since i enables he accele a ion o he mass
69
ans e kine ics o he ime scales o he p ecipi a ion p ocesses. This in ensi ica ion o he
70
p ocess allows an imp o ed con ol o e he p ecipi a ion, a he same ime ha he
71
exposi ion o he p oduc o deg ading high- empe a u e condi ions is dec eased.
72
This wo k p esen s he de elopmen o wa e soluble o mula ions o que ce in h ough
73
p essu ized e hyl ace a e-wa e emulsion echnique. E hyl ace a e has been chosen as o ganic
74
sol en because i is a Gene ally Recognized as Sa e (GRAS) sol en wi h low oxici y (Le hal
75
Dose LD50 in a s: 11.3 g/kg) and i can be sa ely used as a la ou ing agen [17]. Th ee na u al
76
o igin su ac an s, namely modi ied n-oc enyl succina e anhyd ide (OSA) s a ch, soybean
77
leci hin and ba ley β-glucan, we e used in his wo k o he encapsula ion o que ce in. S a ch
78
is he second mos abundan biomass ma e ial p esen in na u e being he mos abundan
79
s o age polysaccha ide in plan s [18,19]. Howe e , na u al s a ch is mainly hyd ophilic, which
80
could limi i s applica ion in he encapsula ion o hyd ophobic compounds. Ne e heless, OSA
81
(modi ied amphiphilic s a ch) is capable o o e come his d awback, and was al eady used o
82
he encapsula ion and deli e y o compounds wi h dis inc pola i ies[19]. Leci hin is a mix u e
83
o na u ally occu ing phospholipids, mainly phospha idylcholine, which is usually a ailable
84
om sou ces such as soybeans o eggs. Phospholipids a e amphiphilic molecules composed by
85
hyd ophobic ails and hyd ophilic heads, being capable o ea ange hemsel es as liposomes,
86
sphe ical and closed s uc u es composed o lipid bilaye s [20,21]. Liposomes a e in e es ing
87
ca ie ma e ials o he deli e y o hyd ophobic/hyd ophilic compounds, and since hey ha e
88
a ini y o cellula memb anes, hey a e capable o inc ease he abso p ion o se e al d ugs
89
[22]. Besides he good p ope ies as encapsula ing agen and emulsi ie , leci hin is also known
90
o i s an ioxidan ac i i y [23]. β-glucans a e soluble ibe s p esen in ce eal g ains, especially
91
in ba ley, cons i u ed by linea polysaccha ides o glucose uni s, connec ed by (1 → 3) o (1 →
92
4)-be a linkages. These ca bohyd a es a e known o hei he apeu ic e ec s on co ona y
93
hea disease, diabe es and hype choles e olemia, and ha e been used as encapsula ing
94
agen s [24–27]. The in luence o he main p ocess pa ame e s has been s udied, namely he
95
e ec o que ce in and emulsi ie ’s concen a ion, he e ec o he lows o o ganic sol en ,
96
suspension o que ce in and dissolu ion o emulsi ie and also he o ganic o wa e a io. By
97
compa ing he esul s ob ained wi h he h ee di e en emulsi ie s, hei oles on he
98
emulsion o ma ion and que ce in’s encapsula ion can be es ablished. P oduc analysis
99
included pa icle size, encapsula ion e iciency, an ioxidan ac i i y and s uc u al
100
cha ac e isa ion.
101
102
2. Ma e ials and me hods
103
2.1. Ma e ials
104
Que ce in hyd a e (Q) wi h a (pu i y≥95%) was pu chased om Sigma-Ald ich. E hyl Ace a e
105
wi h a pu i y o 99.5% and glyce ol we e pu chased om Pan eac Química (Ba celona, Spain).
106
Modi ied OSA-s a ch e ined om waxy maize was kindly p o ided by Na ional S a ch G oup
107
(Hambu g, Ge many). Soybean leci hin (97% phospholipids) was ob ained om Glama-So
108
(SOTYA, Mad id, Spain). Glucagel™ (ba ley β-Glucan) was kindly supplied by DKSH F ance
109
(pu i y 78%, MW : 125 -140 kDa).
110
2.2. P ecipi a ion om p essu ized e hyl ace a e-on-wa e emulsions
111
The equipmen used in his wo k, al eady desc ibed by De Paz e al[15,16], is ep esen ed in
112
Fig. 1 wi h a schema ic low diag am.
113
(FIGURE 1)
114
B ie ly, i consis s o h ee small s o ages a ambien p essu e, co esponding o he eed o
115
pu e e hyl ace a e (V-1), que ce in suspension in he same o ganic sol en (V-2) and he
116
aqueous solu ion o he emulsi ie (V-3). The ins alla ion also coun s wi h wo pis on pumps
117
GILSON 305 (maximum low a e: 25 mL/min; low a e con ol wi h an a accu acy o 0.1
118
mL/min) used o eed he aqueous dissolu ion o he emulsi ie and he que ce in suspension
119
(pumps P-3 and P-2, espec i ely) and a pis on pump JASCO PU-2080 plus (maximum low a e:
120
10 mL/min; low con ol wi h an a accu acy o 0.1 mL/min) used o eed he pu e o ganic
121
sol en (pump P-1). An o en (KNK-2000-C se ies GAS CHROMATOGRAPH) is used o p ehea
122
he o ganic sol en s eam.
123

This p ocess s a s wi h he o al dissolu ion o que ce in in ho and p essu ized e hyl ace a e,
124
whe e by inc easing empe a u e, i is possible o inc ease he solubili y o que ce in in his
125
sol en , which is a ound 1g/L a ambien condi ions. In his wo k, a empe a u e, ypically,
126
be ween 125-140ºC was used, keeping a cons an p essu e be ween 6.0 and 6.5 MPa in o de
127
o main ain he sol en in liquid s a e. The dissolu ion is achie ed by mixing a la onoid’s
128
suspension in p essu ized e hyl ace a e a ambien empe a u e, wi h a s eam o p ehea ed
129
and p essu ized e hyl ace a e, using a T- mixe (M-1 in Fig. 1). In o de o educe he exposu e
130
o que ce in o high empe a u es, his ho and p essu ized solu ion is mixed wi h he
131
ambien - empe a u e aqueous solu ion o emulsi ie using he second T-mixe o he
132
equipmen (M2). The e o e, he con ac ime be ween que ce in and he ho sol en is
133
educed o less han wo seconds, a oiding he deg ada ion o he p oduc . The con ac o he
134
ho solu ion o que ce in wi h he aqueous solu ion in mixe M-2 causes he emulsi ica ion o
135
he o ganic sol en and he p ecipi a ion o que ce in by a combined an isol en and cooling
136
e ec . The las s ep is he emo al o he o ganic sol en om he o med emulsion using a
137
o a y e apo a o , in o de o ob ain an aqueous dispe sion o que ce in pa icles s abilized by
138
he su ac an .
139
A ypical expe imen s a ed wi h he p epa a ion o an aqueous solu ion o emulsi ie and a
140
suspension o que ce in in e hyl ace a e, which we e p e-hea ed and s i ed in o de o ob ain
141
homogeneous mix u es. A e wa ds, pumps we e swi ched on and he p essu e in he sys em
142
was ixed be ween 6.0 and 6.5 MPa. The o en was swi ched on o hea he o ganic sol en
143
un il he desi ed empe a u e (140-150ºC). When he equi ed empe a u e in mixe M-1 was
144
eached, he pu e sol en s used un il his momen we e eplaced wi h he que ce in
145
suspension, in con inuous agi a ion, and wi h he aqueous solu ion o he emulsi ie . In all
146
expe imen s, a olume o app oxima ely 25 mL o suspension con aining que ce in was
147
p ocessed. A e wa ds, he o ganic sol en o he ob ained emulsion was emo ed om he
148
sample using a o a y e apo a o (BÜCHI 011-BÜCHI 461 Wa e Ba h) unde acuum (-0.06,
149
60ºC and 110min), o elimina e he o ganic sol en .
150
2.3. P oduc cha ac e iza ion
151
2.3.1. Mic oscopy
152
An au oma ed up igh mic oscope sys em o li e science esea ch (Leica DM4000 B, We zla ,
153
Ge many) was used o ob ain mic oscopic images o he emulsions and suspensions.
154
2.3.2. Physical s abili y o emulsion
155
The physical s abili y o p epa ed emulsions was s udied h ough he op ical analyse
156
Tu biscan™ Classic on he p oduc ion day, by measu ing he a ia ions in backsca e ing.
157
2.3.3. Pa icle size
158
The pa icle size o he micelles we e measu ed by a Lase Di ac ion (LD) equipmen model
159
Mal e n Mas e size 2000 ollowing a cen i uga ion s ep a 7800 pm, 5min, in o de o
160
p ecipi a e he c ys als o que ce in ha we e no encapsula ed. Suspensions we e dispe sed
161
in dis illed wa e and measu emen was ca ied ou a e a gen le o a ion o he suspension
162
con aine in o de o ob ain a be e dispe sion o he micelles. In his wo k, pa icle size
163
measu emen s a e epo ed as olume dis ibu ion and de ined as he a e age diame e (d0.5).
164
2.3.4. Encapsula ion e iciency (EE)
165
Fo he que ce in’s wa e suspensions s abilized wi h s a ch he Encapsula ion E iciency (EE)
166
was de e mined h ough Ul a iole - isible spec opho ome y, while in he suspensions
167
s abilized wi h leci hin o β-glucan, due o he in e e ence o hese ca ie ma e ials on UV
168
assays, his pa ame e was de e mined by HPLC.
169
Fo UV-Vis measu emen s, he suspensions we e p e iously cen i uga ed a 7800 pm (5min)
170
and dilu ed in wa e . The concen a ion o que ce in was de e mined using a UV-Visible
171
Spec opho ome e (Shimadzu UV-2550, λ= 375 nm). Calib a ion was ob ained by using
172
s anda d samples wi h concen a ions be ween 3 and 12μg/mL, using e hanol as sol en .
173
HPLC analysis o que ce in was pe o med in acco dance wi h a me hod p e iously epo ed
174
[28]. A Wa e s 515 HPLC pump, equipped wi h In-Line Degasse AF (Wa e s), 717 plus
175
Au osample (Wa e s) and 2487 Dual ʎ Abso bance de ec o (Wa e s) we e used.
176
Ch oma og aphic sepa a ion was ca ied ou wi h a Symme y C18 Column (5 µm, 4.6 mm X
177
150 mm, Wa e s) coupled wi h a Bio-Sil C18 p ecolumn (5 µm, 4.6 mm X 30 mm, Bio-Rad) wi h
178
mobile phase o ace oni ile/5% ace ic acid solu ion (27:73, / ) a 30 ◦C. The de ec ion
179
wa eleng h was se a 373 nm and he low a e was 1.0 mL/min. The calib a ion cu e was
180
linea wi hin he ange o 25–200µg/mL (R2= 0.9997) using me hanol:wa e (70:30) solu ion as
181
sol en . The que ce in suspension samples we e de ec ed by HPLC ollowing cen i uga ion a
182
7800 pm (5min) and dilu ion in me hanol. The un ime o he assay was 13 min and he
183
e en ion ime o que ce in was 8.32 min.
184
The abso bance is p opo ional o he amoun o que ce in dispe sed in solu ion which
185
co esponds o he encapsula ed que ce in. Non-encapsula ed que ce in can co espond
186
ei he o que ce in p ecipi a ed as big c ys als and no s abilized in he suspension, o o
187
que ce in ha has unde gone deg ada ion du ing he p ocess. The encapsula ion e iciency is
188
epo ed as he a io be ween he amoun o la onoid dispe sed in solu ion and o al
189
que ce in amoun in he ini ial eed.
190
2.3.5. S uc u al cha ac e isa ion
191
In a ed spec a o he suspensions and pu e ma e ials we e eco ded on a B uke ALPHA FT-IR
192
appa a us equipped wi h a Pla inum ATR sampling module including a diamond c ys al. The
193
suspensions we e cen i uged a 13300 pm du ing 30min. The supe na an was emo ed and
194
he sample pelle ’s was submi ed o u he e apo a ion o wa e a -0.09MPa and 35ºC o
195
48h. The pelle was hen used o pe o m FTIR. The spec a in he ange om 4000 o 400 cm-1
196
we e he a e age o 64 scans a a esolu ion o 2cm-1. The ATR signal was ans o med o
197
T ansmi ance and he ob ained spec a was no malised a e he baseline co ec ion.
198
2.3.6. C yo-TEM
199
C yo-TEM analysis we e pe o med h ough a me hod al eady desc ibed [29]. B ie ly, 4μl o
200
sample was deposi ed on a ack C-Fla 1.2/1.3, which was p e iously hyd ophilized by a plasma
201
cleane . A blo ing is pe o med on ei he side o he il e pape g id du ing 4s whe e,
202
a e wa ds, liquid e hane is in oduced in o de o eeze he samples, a oiding he o ma ion
203
o c ys als (Ga an C yoplunge 3). Samples a e main ained in liquid ni ogen un il hei ans e
204
o he holde (Ga an C yo ans e 626). JEOL JEM-FS2200 HRP 200 kV TEM equipmen wi h
205
elec on il e ing was used o pe o m he TEM images.
206
2.3.7. S abili y o suspensions
207
One o he aqueous suspensions wi h he bes ela ion be ween EE and inal que ce in’s
208
concen a ion was chosen o s abili y examina ion. Some au ho s demons a ed ha i ’s
209
possible o inc ease he s abili y o leci hin, educing he appea ance o i s agglome a es and
210
inc ease d ug’s solubili y by adding glyce ol (G), as co-sol en , o aqueous leci hin dispe sions
211
[30,31]. Thus, an equal suspension wi h 2.5% ( / ) o glyce ol as co-sol en in he wa e phase
212
was p epa ed in o de o compa e he physicochemical p ope ies o he suspension wi hou
213
i . The s abili y examina ion was pe o med h ough he assessemen o micella pa icle size,
214
p esence o c ys als, que ce in’s concen a ion and an ioxidan ac i i y o he suspensions
215
a e 75 days o s o age in a e ige a ed da k oom (10ºC).
216
The assessmen o an ioxidan ac i i y was pe o med h ough oxygen adical abso p ion
217
capaci y (ORAC) assay, which was ca ied ou by a me hod p e iously desc ibed [32]. This assay
218
measu es he abili y o he an ioxidan species p esen in he sample o inhibi he oxida ion o
219
disodium luo escein (FL) ca alysed by pe oxyl adicals gene a ed om AAPH.
220
impo an o emembe ha u biscan only p o ides mean pa icle size alue bu no he
369
dispe sion o his alue, hence he di e ences be ween u biscan’s alues and op ical
370
mic oscope images.
371
Rega ding expe imen E20, c ys als o que ce in p ecipi a ed in he lask du ing he
372
e apo a ion o he emulsions, which was con i med by op ical mic oscopy. β-Glucan did no
373
seem o be a good ca ie /emulsi ie o encapsula e his la onoid, and so, highe
374
concen a ions o que ce in we e no es ed. The inal pa icle o he que ce in-loaded
375
micelles was in he mic ome e ange. In he case o E20, he p esence o que ce in’s c ys als
376
con ibu ed o he mic ome e size, whe eas in he samples E21-E23 he p esence o ca ie ’s
377
agglome a es con ibu ed o his pa icle size. Compa ing E20 and E22, i is possible o see ha
378
he addi ion o leci hin inc eased he pa icle size due o he o ma ion o leci hin:β-glucan
379
complexes [41]. Ne e heless, he addi ion o mo e leci hin (E21 and E23) led o a educ ion o
380
pa icle size, p obably due o he o ma ion o highe amoun o small sized que ce in
381
encapsula ed pa icles.
382
Rega ding he encapsula ion e iciency, i is possible o e i y ha compa ing E20 and E22, he
383
addi ion o leci hin o he sys em led o a dec ease o EE. The addi ion o leci hin led o he
384
complexa ion o he ca ie s, and hei u he p ecipi a ion. Pe haps he e is a compe i ion
385
be ween que ce in and leci hin o he complexa ion wi h β-glucan, esul ing in less EE in E22
386
compa ing wi h E20.
387
Ne e heless, as he amoun o he leci hin inc eased in β-glucan:leci hin mix u es
388
(E22<E21<E23), so as inc eased he EE o he sys em. These alues a e in ag eemen wi h he
389
educ ion o pa icle size in hese samples, as discussed p e iously. Pe haps in expe imen E23
390
he e is a highe po ion o ee leci hin, no complexed wi h β-glucan, o in e ac wi h
391
que ce in, inc easing i s encapsula ion. This ac ein o ces he capaci y o leci hin o
392
encapsula e que ce in
393
3.4. S abili y
394

Expe imen E17 was epea ed wi h and wi hou he inclusion o glyce ol in o de o e alua e
395
and compa e hei s abili y. Fo ha pu pose, he micella pa icle size, p esence o c ys als,
396
que ce in’s concen a ion and an ioxidan ac i i y o he esul ing suspensions we e measu ed
397
a e 75 days o s o age. Du ing his pe iod, he aqueous solu ions we e s o ed in a
398
e ige a ed oom a empe a u es a ound 10ºC and p o ec ed om ligh . The e ige a ion
399
condi ion was chosen o he s abili y s udy since i is ecommended as he s o age condi ion
400
o he phospholipids [42]. Resul s a e epo ed in Table 4.
401
(TABLE 4)
402
As p esen ed in his able, he sample p epa ed wi h glyce ol as co-sol en main ained
403
colloidal s abili y a he end o one mon h and a hal , wi h no signi ican change in i s pa icle
404
size, indica ing good s abili y du ing his pe iod. In con as , he sample wi hou glyce ol nea ly
405
ipled i s size a e he same pe iod, being his alue e en highe a e wo mon hs and a hal
406
a e i s p epa a ion.
407
(FIGURE 6)
408
E alua ing he shape o he size dis ibu ion, shown in Fig. 6, he dis ibu ion o he sample
409
eshly p epa ed ha e main peak in he nanome e s ange, co esponding o que ce in
410
pa icles ha we e success ully encapsula ed, and o he peaks a sizes abo e 1 µm possibly
411
co esponding o que ce in’s c ys als and leci hin’s agglome a es. Du ing s o age, he peaks
412
abo e 1 µm a e displaced o conside ably highe pa icle sizes, indica ing ha hese bigge
413
pa icles a e becoming mo e agglome a ed and u he des abilizing he suspension due o
414
hei highe size. The addi ion o a co-sol en is essen ial o an adequa e s abili y o he
415
o mula ion, a oiding no only he agglome a ion o leci hin, bu also delaying he appea ance
416
o que ce in’s c ys als. In ac , in he sample p epa ed wi hou glyce ol, he appea ance o
417
la onoid’s c ys als was obse ed sho ly a e i een days o p epa a ion. Rega ding
418
encapsula ion e iciency, he addi ion o a co-sol en appea ed o sligh ly enhance he
419
solubili y o que ce in in aqueous media. Ne e heless, he s abili y o que ce in o e s o age
420
was simila in he wo es ed samples.
421
Table 4 also shows he esul s achie ed by ORAC an ioxidan assay o he wo suspensions
422
o e s o age. The esul s a e exp essed as mic omoles o T olox equi alen s pe g am o
423
que ce in. An ioxidan ac i i y o pu e que ce in a e leci hin was also measu ed in his wo k,
424
e ealing alues o 6586 and 2.5 μmol o TE/g o p oduc , espec i ely. The p obable o ma ion
425
o hyd ogen bonds be ween leci hin and que ce in esul ed in a 3- old inc eased an ioxidan
426
ac i i y pe uni mass o que ce in o bo h suspensions, being hese alues nea ly p ese ed
427
a e 75 days o s o age.
428
4. Conclusion
429
The o mula ion o que ce in using h ee dis inc emulsi ie s, namely n-oc enyl succina e
430
anhyd ide s a ch, soybean leci hin and ba ley β-glucan, was in es iga ed in his wo k h ough
431
p essu ized e hyl ace a e-in-wa e emulsions. Resul s showed ha OSA-s a ch was no sui able
432
o he encapsula ion o que ce in due o he low encapsula ing e iciencies achie ed (below
433
10%) and micella pa icle sizes in he ange o mic ome e s. Expe imen s ca ied ou wi h
434
leci hin yielded he bes esul s, wi h encapsula ion e iciencies up o nea 76% and pa icle
435
sizes in he sub-mic ome e ange. Mo eo e , he aqueous suspensions o que ce in s abilized
436
wi h his su ac an p esen ed concen a ions o he la onoid up o 630ppm, being 315-
437
old highe compa ed wi h solubili y o pu e que ce in in wa e . The o ma ion o
438
hyd ogen bonds be ween leci hin and que ce in could explain he good esul s ob ained
439
wi h his emulsi ie , u he comp ising imp o ed an ioxidan ac i i y (3- old highe pe
440
uni mass o que ce in) due o a syne gis ic e ec . Fu he mo e, he addi ion o leci hin o
441
he emulsions s abilized wi h β-glucan imp o ed he encapsula ion e iciency and pa icle
442
size o he micelles, ein o cing he capaci y o leci hin o encapsula e que ce in.
443
The esul s ob ained in his wo k showed ha high p essu e emulsion echnique is a e sa ile
444
me hod o he p oduc ion o wa e soluble o mula ions o que ce in, a oiding que ce in’s
445
deg ada ion and inc easing i s solubili y. Fu he mo e, he addi ion o glyce ol as co-sol en
446
can inc ease he colloidal s abili y o he suspension and he encapsula ion e iciency o
447
he la onoid.
448
Acknowledgemen s
449
This wo k was suppo ed by Ma ie Cu ie Indus y-Academia Pa ne ships and Pa hways
450
(Eu opean Commission) h ough he WineSense p ojec (FP7-PEOPLE-2012- IAPP-612608).
451
V.S.S. Gonçal es is g a e ul o he doc o al g an SFRH/BD/77350/2011 om Fundação
452
pa a a Ciência e Tecnologia. S. Rod íguez-Rojo acknowledges he Minis e io de Economía y
453
Compe i i idad and Uni e sidad de Valladolid o he Juan de la Cie a ellowship (JCI-
454
2012-14992).
455
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