Production of water soluble quercetin formulations by pressurized ethyl acetate-in-water emulsion technique using natural origin surfactants

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. 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