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BARLEY AND YEAST β-GLUCANS AS NEW EMULSIFIER AGENTS FOR THE DEVELOPMENT OF
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AQUEOUS NATURAL ANTIFUNGAL FORMULATIONS
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Ma a Salgado, So aya Rod íguez-Rojo*, Ma ía José Coce o
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High P essu e P ocesses G oup, Depa men o Chemical Enginee ing and En i onmen al
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Technology, EII Sede Me gelina, Uni e si y o Valladolid, 47011 Valladolid, Spain.
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* Co esponding au ho a : So aya Rod íguez-Rojo. Tel: +34 983 423166. E-mail ad ess:
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[email p o ec ed] a.es
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ABSTRACT
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Ba ley and yeas β-glucans we e selec ed, oge he wi h leci hin, o encapsula e es e a ol by
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emulsi ica ion-e apo a ion me hod o de elop new and sa e an i ungal o mula ions. Di e en
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emulsi ica ion echniques we e used: high-shea , high p essu e and high p essu e and
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empe a u e emulsi ica ion. Mo phology, c ys allini y, encapsula ion e iciency and in i o
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an i ungal ac i i y agains Bo y is cine ea o he di e en o mula ions we e e alua ed. No
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signi ican di e ences be ween each emulsi ica ion p ocedu e in pa icle size (below 90 nm) and
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in encapsula ion e iciency (70-100%) we e obse ed; only ba ley β-glucan emulsions showed
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lowe e iciency due o he o ma ion o a gel ha e ained mos o he ac i e compound. A
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g ea in luence o he emulsi ica ion me hod and he encapsula ing ma e ial on he c ys allini y
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o he pa icles was obse ed. The highes an i ungal ac i i y (up o 53% g ow h inhibi ion) was
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ob ained by he o mula ions wi h yeas β-glucans, indica ing an enhanced abso p ion o
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encapsula ed es e a ol h ough he cell wall o he ungus a he p esence o (1-3, 1-6)-β-
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glucans.
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Keywo ds: β-glucan, leci hin, es e a ol, encapsula ion, high-p essu e emulsi ica ion, Bo y is
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cine ea.
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1. In oduc ion
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Fungal and bac e ial in ec ions a e a majo conce n in ag icul u e since hey p oduce g ea losses
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(Spada o & Gullino, 2004). On one hand, many ui s and ege ables mus be disca ded because
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hey canno be eco e ed once hey a e a ec ed, aising an e hical issue due o he inc easing
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global demand o ood de i ed om a bigge popula ion (Bebbe & Gu , 2015). On he o he
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hand, p oduce s mus ca y ou a big in es men in o de o keep in ec ions unde con ol and
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o minimize he damages on hei c ops. The e o e, i is impo an o de elop e ec i e p oduc s
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which educe he g ow h o pa hogens and p ese e he quali y o ood. Fo many yea s, se e al
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chemical p oduc s ha e been used wi h his pu pose. Howe e , hey can lea e oxic esidues on
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he plan , which a e wa ds could a ec human heal h and con amina e he en i onmen (C uz
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Cab al, Fe nández Pin o, & Pa ia ca, 2013). Fu he mo e, ungi and bac e ia can de elop
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esis an s ains a e p olonged applica ion (Panebianco e al., 2015). Thus, in ecen yea s
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he e is a endency owa ds he de elopmen o al e na i e an i ungal and an ibac e ial
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p oduc s om non- oxic, na u al o igin subs ances (Ma i, Be olini, & P a ella, 2003).
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In his sense, essen ial oils ha e been ex ensi ely s udied because o hei an i ungal p ope ies
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(Soylu, Ku , & Soylu, 2010; S e ić e al., 2014). The biocide ac i i y o essen ial oils is mainly due
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o hei con en in polyphenolic compounds ha inc eases cell memb ane pe meabili y and
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p omo es i s dis up ion (Tiwa i e al., 2009); hey also igge de ense mechanisms in he
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in ec ed plan : p oduce alkaliniza ion o he medium, s imula e oxida i e bu s and induce
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de ense genes (Chang, Heene, Qiao, & Nick, 2011). The e o e, many wo ks analyze plan
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ex ac s, ocusing on hei phenolic con en and hei ac i i y agains di e en ungi, like
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Aspe gillus, Penicillium, Bo y is o Candida (Ga o e al., 2011; Ma ins, Ba os, Hen iques, Sil a,
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& Fe ei a, 2015; Rashed, Ći ić, Glamočlija, & Soko ić, 2014).
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The aim o his wo k is o de elop a liquid an i ungal o mula ion wi h a model phenolic
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compound, es e a ol, om an oil-in-wa e emulsion and emo al o he o ganic sol en .
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Res e a ol is included among phy oalexins, seconda y me aboli es ha a e syn hesized by
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plan s as p o ec i e agen s agains bac e ial and ungal a acks (Ad ian & Jeande , 2012).
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Res e a ol is p esen in many plan s, al hough g apes ha e he highes concen a ion
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(Fe nández-Ma , Ma eos, Ga cía-Pa illa, Pue as, & Can os-Villa , 2012). An i ungal ac i i y o
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es e a ol has been p e iously epo ed (Jung e al., 2005), also agains Bo y is cine ea (Ad ian,
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Jeande , Veneau, Wes on, & Bessis, 1997), which is a widely ex ended ungus ha a ec s se e al
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ui s and plan s in pos ha es s age, causing g ea losses (Williamson, Tudzynski, Tudzynski, &
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Van Kan, 2007). Besides, s uc u al changes in conidia upon es e a ol applica ion we e
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obse ed (dis up ed plasma memb ane, diso ganized cy oplasm wi hd awn om conidial wall),
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which could explain he mode o ac ion o es e a ol leading o cell dea h (Ad ian & Jeande ,
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2012). Howe e , ungal laccases p oduce ex acellula oxida ion o es e a ol and i s
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deg ada ion (Chang e al., 2011; Ri e a-Hoyos e al., 2013; Timpe io, D’Alessand o, Fagioni,
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Mag o, & Zolla, 2012). The e o e, a p ope o mula ion is equi ed in o de o encapsula e
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es e a ol and p o ec i om laccases un il he a ge . In ou p e ious wo k, solid pa icles o
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es e a ol encapsula ed on ba ley β-glucans we e ob ained by emulsi ica ion-e apo a ion and
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u he d ying o he suspensions (Salgado, Rod íguez-Rojo, Al es-San os, & Coce o, 2015).
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These pa icles showed an i ungal ac i i y agains B. cine ea. Howe e , a liquid o mula ion
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would be p e e ed because i is easie o apply in si u. Res e a ol has been p e iously
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o mula ed as liquid suspension in di e en ma e ials such as lipids, and syn he ic and na u al
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polyme s (p o eins and polysaccha ides), mainly o pha maceu ic, cosme ic o ood- ela ed
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applica ions. These suspensions we e c ea ed by di e en me hods, including e apo a ion
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(Puja a, Jambh unka , Wong, McGuckin, & Popa , 2017), an isol en p ecipi a ion and
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elec os a ic deposi ion (Xulin Huang e al., 2017), ionic gela ion (Jeon, Lee, & Lee, 2016), o
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ul asounds (Caddeo e al., 2016), among o he s.
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Lipids (e.g. a y acids, leci hin) a e commonly used as su ac an s because hey can inco po a e
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bo h hyd ophilic and lipophilic ac i e compounds, a e non- oxic and easily abso bed h ough
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biological memb anes due o he o ma ion o liposomes, which a e e y simila o cell
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memb anes (Va ona e al., 2013). Polysaccha ides a e also used because hey enhance he
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s abili y o he emulsion h ough an inc ease o iscosi y ha educes he mo emen o he
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d ople s wi hin he emulsion (Ga cía, Al a o, Cale o, & Muñoz, 2014). Among polysaccha ides,
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β-glucans, polyme s o D-glucose linked by glycosidic bonds in posi ions (1-3), ha e been
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selec ed because hey a e p esen in se e al ungi, bac e ia and some kind o plan s, including
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ba ley o oa . The e o e, i is hypo hesized ha , since β-glucans a e p esen in he cell wall o B.
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cine ea (Tenbe ge, 2007), hey may imp o e he abso p ion o he encapsula ed ac i e
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compound and hus inc ease i s e ec i eness, besides ac ing as su ac an . β-glucans ha e
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di e en s uc u e depending on hei o igin. Fo ins ance, ce eal β-glucans ha e (1-4)
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b anching, whe eas yeas β-glucans (YBG) ha e (1-6) b anching (Zhu, Du, & Xu, 2016). Also, he
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a ia ion on hei s uc u e p o ides hem di e en physical and biological p ope ies. Fo
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ins ance, b anched (1-3),(1-6)-β-glucans ha e s onge immune modula o y p ope ies han (1-
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3),(1-4)-β-glucans (Mikkelsen, Jespe sen, Mehlsen, Engelsen, & F økiæ , 2014). Mo eo e , many
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wo ks epo he abili y o β-glucans as elici o s in plan s o induce he o ma ion o seconda y
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me aboli es and igge de ense esponses (Almag o, Ga cía-Pé ez, Belchí-Na a o, Sánchez-
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Pujal e, & Ped eño, 2016; Aziz e al., 2003).
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β-glucans ha e been used as encapsula ing ma e ial o an hocyanins by coace a ion (Xiong,
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Mel on, Eas eal, & Siew, 2006), o p o eins h ough he p oduc ion o c yogels (Laza idou,
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K i ikopoulou, & Biliade is, 2015) and o es e a ol by sp ay-d ying and PGSS-d ying (Salgado
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e al., 2015). In emulsion, hey ha e been p e iously used as s abilize s (Bu kus & Temelli, 2000;
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Kon ogio gos, Biliade is, Kiosseoglou, & Doxas akis, 2004; Thammaki i, Suphan ha ika,
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Phaesuwan, & Ve duyn, 2004). Ba ley β-glucans (BBG) we e ound o educe su ace ension in
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aqueous solu ions, eaching a cons an alue o 50-55 mN/m a 2 g/L (unpublished wo k).
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Howe e , o he au ho s’ knowledge, he e is jus one wo k in which hey we e used as
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su ac an o emulsions, bu i was ound ou ha he ac i e compound (que ce in) p ecipi a ed
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in c ys als inside he polyme , so o a oid his, leci hin was added as co-su ac an (Gonçal es e
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al., 2015).
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In his wo k, β-glucans we e used as encapsula ing ma e ial o he p oduc ion o liquid
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o mula ions o es e a ol by emulsi ica ion-e apo a ion. Also leci hin was used, bo h alone
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and mixed wi h he β-glucans, in o de o enhance he o ma ion o he emulsion. Di e en
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emulsion echniques we e used: high-shea , high p essu e and high p essu e and empe a u e
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emulsi ica ion. The la e me hods we e pe o med because hey we e epo ed o p o ide
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highe encapsula ion e iciencies (de Paz, Ma ín, Ma eos, & Coce o, 2013). Finally, he
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an i ungal ac i i y o he o mula ions wi h es e a ol was es ed agains B. cine ea.
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2. Ma e ials and me hods
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2.1. Ma e ials
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As encapsula ing ma e ials, wo di e en ypes o β-glucans we e used: ba ley (1-3, 1-4)-β-
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glucans (75% pu i y; 125 kDa, de e mined as indica ed in (Salgado e al., 2015) Glucagel, kindly
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supplied by DKSH, F ance) and (1-3, 1-6)-β-glucans om yeas Saccha omyces ce e isae (64%
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pu i y, de e mined as indica ed in sec ion 2.2.1. β-glucan con en ; L-Na u ae Nu aceu ical,
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kindly supplied by Na u ae, Spain). Soybean leci hin (Glama-so , SOTYA S.A., Spain) was also
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used as encapsula ing ma e ial. E hyl ace a e (99%), mal ex ac aga , ace ic acid glacial and
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sodium ace a e we e pu chased om Pan eac (Spain). Res e a ol wi h 98% pu i y was
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pu chased om Pu e Bulk (USA). Pullulan s anda ds we e pu chased om Shodex.
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2.2. Solubiliza ion and cha ac e iza ion o YBG
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BBG we e soluble in wa e , so hey could be di ec ly dissol ed in wa e and used in
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emulsi ica ion. Howe e , YBG we e insoluble in wa e , he e o e a p e ea men was equi ed
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in o de o solubilize hem. Fo ha pu pose, a 170 mL s ainless s eel essel was used. Beside
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YBG, sodium ace a e (1.41 mg/g o β-glucan) and glacial ace ic acid (12.2 μL/g o β-glucan) we e
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cha ged in o he essel a concen a ions epo ed in (Cox, 2008), wi h a solid o liquid a io o
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1:10 (w:w). The essel was hea ed wi h an elec ic ex e nal esis ance and kep a 135° C and
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0.28 MPa o 4.5 hou s. Samples o he p oduc we e ob ained a 3, 3.5, 4 and 4.5 h. Fu he
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desc ip ion o he equipmen can be ound in (Sánchez-Bas a do, Rome o, & Alonso, 2017) .
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2.2.1. β-glucan con en
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The quan i y o β-glucan on he solubilized YBG was analyzed by means o he “β-Glucan Assay
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Ki (Yeas & Mush oom)” om Megazyme (I eland). Following he desc ibed p ocedu e in he
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ki , he pe cen age o o al glucans and α-glucans was de e mined spec opho ome ically
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(Shimadzu UV-2550), and he pe cen age o β-glucans was calcula ed as he di e ence be ween
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he o al and he α-glucans.
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2.2.2. Molecula weigh o he β-glucans
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Molecula weigh o YBG was measu ed by size exclusion ch oma og aphy, wi h a gua d column
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(Shodex SB-G), a column (Shodex SB-804 HQ, pa icle size 10 μm, 7.8x300 mm) and a di e en ial
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e ac i e index de ec o (410, Wa e s Co po a ion). The column was kep a 35º C and low a e
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o he mobile phase (0.1 M NaNO3 and 0.02% NaN3) was se a 0.4 mL/min. Pullulan s anda ds
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(5.9-708 kDa) we e used.
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2.3. P epa a ion o emulsions
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2.3.1. High shea emulsi ica ion
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An oil-in-wa e emulsion was o med, con aining es e a ol dissol ed in e hyl ace a e (7.5 g/L)
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as o ganic phase and he encapsula ing ma e ial dissol ed in he aqueous phase, p e iously
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sa u a ed wi h e hyl ace a e. Di e en concen a ions we e es ed in he aqueous phase (10, 15
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and 20 g/L) o each o he encapsula ing ma e ials used: BBG, solubilized YBG and leci hin, and
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mix u es o leci hin and each o he glucans (50% in weigh ).
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Bo h liquid phases we e mixed in a a io 1:9 ( : ) a 800 pm o 5 min and hen he emulsion
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was ed in o a 200 mL o o -s a o machine (IKA LABOR PILOT 2000/4) and p ocessed o 4
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minu es a 4200 pm, as desc ibed in (Salgado e al., 2015).
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2.3.2. P ecipi a ion om a p essu ized emulsion
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High p essu e emulsi ica ion was also pe o med wi h he same aqueous and o ganic phases
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a o emen ioned, a 6 MPa and ambien empe a u e. The aqueous solu ion was p essu ized
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wi h a Dosap o Mil on Royal D (18 mL/min, 20 g su ac an /L), whe eas a Jasco PU-2080 plus
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pump was used o eeding he o ganic phase (2 mL/min, 7.5 g es e a ol/L). A e
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p essu iza ion, bo h s eams we e pu in con ac in a T-mixe , whe e he emulsion was o med,
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and a e wa ds he p oduc was eco e ed.
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2.3.3. P ecipi a ion om a ho p essu ized emulsion
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This me hod o encapsula ion was p e iously used (de Paz e al., 2012) o accele a e he mass
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ans e kine ics o he ime scales o he p ecipi a ion p ocess, hus imp o ing he con ol o e
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he p ecipi a ion. B ie ly, a solu ion o suspension o he ac i e compound in he o ganic phase
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is pu in con ac wi h ano he s eam o pu e o ganic sol en a high empe a u e and
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immedia ely a e wa ds his low is mixed wi h he cold wa e phase which con ains he
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su ac an . This causes he emulsi ica ion o he o ganic phase and he p ecipi a ion o he ac i e
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compound in o he polyme . The emulsion is hus o med by he combined an i-sol en and
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cooling e ec o he wa e , and he ac i e compound does no unde go he mal deg ada ion
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du ing he p ocess because due o he con igu a ion o he equipmen , he exposi ion ime o
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high empe a u e is lowe han 2 seconds. A de ailed explana ion abou he equipmen used
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can be ound in (de Paz e al., 2012).
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The ope a ion s a s wi h he p essu iza ion o he 3 s eams up o 6.0-6.5 MPa and hea ing up
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o he o ganic sol en o he desi ed empe a u e (in his case, 85-90ºC). When he ope a ing
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condi ions we e eached, he eed was changed om pu e wa e and e hyl ace a e o he
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solu ions used in his wo k: a solu ion o e hyl ace a e wi h 7.5 g es e a ol/L a 2.75 mL/min,
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ho e hyl ace a e a 4.25 mL/min and a solu ion o 20 g su ac an /L in wa e a 20 mL/min. The
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ope a ing condi ions we e selec ed based on a p e ious wo k in which que ce in was
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encapsula ed on s a ch, leci hin and β-glucans (Gonçal es e al., 2015).
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2.4. Fo ma ion o suspensions
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Suspensions we e ob ained o all he di e en emulsi ica ion me hods by emo ing he o ganic
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sol en by acuum e apo a ion (Heidolph) a 60º C, 75 pm and a acuum o 0.08 MPa.
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A e wa ds, big agglome a es o polyme and non-encapsula ed c ys als o es e a ol we e
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emo ed by cen i uga ion a 7800 pm (6461 g) o 10 min.
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2.5. Cha ac e iza ion o emulsions and suspensions
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2.5.1. D ople size dis ibu ion
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D ople and pa icle size dis ibu ion o he emulsions and suspensions was measu ed by lase
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di ac ion (Mas e size 2000, Mal e n) by dilu ing he emulsions on a solu ion o wa e
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sa u a ed wi h e hyl ace a e and he suspensions only in wa e . Pa icle size was measu ed bo h
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be o e and a e cen i uga ion o he suspensions. Resul s a e exp essed as % in numbe . The
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d ople and pa icle sizes epo ed co espond o he alue o d(0.5).
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2.5.2. C yoTEM
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C yoTEM analysis o he aqueous solu ions o he di e en β-glucans and hei mix u es wi h
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leci hin we e pe o med o check how hei chains associa ed hemsel es, in a JEOL JEM-FS2200
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HRP 200 kV TEM wi h elec on il e ing. In b ie , 4 μL o sample was deposi ed on a ack C-Fla
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1.2/1.3, which was p e iously hyd ophilized by a plasma cleane . A blo ing is pe o med on
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ei he side o he il e g id du ing 4 s and hen liquid e hane is in oduced he e in o de o
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eeze he samples, a oiding he o ma ion o c ys als (Ga an C yoplunge 3). Samples a e
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main ained in liquid ni ogen un il hei ans e o he holde (Ga an C yo ans e 626).
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2.5.3. X- ay di ac ion
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X- ay di ac ion (XRD) measu emen s we e pe o med on a B uke Disco e D8 di ac ome e
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o check he c ys allini y o he pa icles in he inal suspensions, using he Cu Kα adia ion (λ =
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0.15406 nm). The suspensions we e u he cen i uged a 20000 pm o 30 min (Beckman) in
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o de o eco e he encapsula ed pa icles. A e wa ds, his sedimen was d ied in a eeze-
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d ie (LyoQues -55, Tels a ) o 48 hou s and he inal powde was analyzed by XRD. The
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sca e ing in ensi ies we e measu ed o e an angula ange o 5 < 2θ < 70 o all he samples,
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wi h a s ep size o 0.02°.
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2.5.4. Encapsula ion e iciency
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Quan i ica ion o encapsula ed es e a ol was done by HPLC wi h a gua d column (Bio-Sil C18
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HL 90-5, 4.6 x 30 mm, pa icle size 5 µm, Bio-Rad), a column (Symme y C18, 4.6 x 150 mm,
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pa icle size 5 µm, Wa e s) and a UV de ec o (λ = 306 nm). The column was kep a 25º C and
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low a e o he mobile phase (ace oni ile and wa e in a io 1:3, wi h 0.2% o mic acid) was se
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a 0.8 mL/min. Encapsula ion e iciency was de e mined as he a io be ween he quan i y o
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es e a ol in he cen i uged suspension and he amoun o es e a ol in he ini ial emulsion.
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Calib a ion ange o es e a ol was be ween 10 and 40 ppm. P io o analysis, he cen i uged
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suspensions we e dilu ed (0.6 mL o sample in 10 mL) and il e ed (0.22 μm). The analysis was
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pe o med in iplica e, and he da a we e analyzed by -S uden ´s es (unpai ed samples,
216
unequal a iances) wi h a signi icance p- alue o 0.05.
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2.6. B. cine ea cul u e
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Fo he in i o cul u e o B. cine ea, he ungus was isola ed om ines in ou uni e si y (Campus
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La Yu e a, Uni e sidad de Valladolid, Palencia, Spain) and i was g own on mal ex ac aga .
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A e au ocla ing, he aga was pou ed in o Pe i dishes con aining he co esponding sample o
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con ol, and when i solidi ied, he ungus was placed in he cen e o he su ace. The quan i y
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o sample on each pla e was de e mined so as o ha e a concen a ion o es e a ol o 100
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Encapsula ion e iciency was much lowe wi h BBG because du ing cen i uga ion a gel-like
334
s uc u e was o med and i e ained mos o he compounds. No signi ican di e ences we e
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obse ed be ween each emulsi ica ion me hod wi h any o he encapsula ing ma e ials. In some
336
p e ious wo ks much highe encapsula ion e iciencies we e achie ed by high p essu e and
337
empe a u e emulsi ica ion (80%) han by high-shea emulsi ica ion (8%) in he o mula ion o
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β-ca o ene wi h modi ied s a ch (de Paz e al., 2013). Howe e , in ha case he inc ease was
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due o he highe solubili y o he ac i e compound in he ho o ganic sol en , whe eas in his
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wo k he concen a ion o es e a ol in he o ganic phase was kep cons an in all he
341
expe imen s.
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3.3. B. cine ea cul u e
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Fo he samples by high-shea emulsi ica ion, no signi ican di e ences we e obse ed be ween
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each p oduc a he lowes concen a ions o su ac an (10 and 15 g/L), since none o hem had
345
e ec on he ungal g ow h ( esul s no shown). Howe e , some inhibi ion was achie ed wi h
346
he suspensions a 20 g/L o encapsula ing ma e ial, also o he samples by high-p essu e and
347
high-p essu e and empe a u e emulsi ica ion. Figu e 6 p esen s ungal g ow h a ea when
348
applying he suspensions o es e a ol o mula ed wi h leci hin (Lec), a mix u e o leci hin and
349
BBG (LecBBG), BBG, a mix u e o leci hin and YBG (LecYBG) and YBG. The esul s shown he e
350
co espond o he cen i uged suspensions excep in he case o BBG. These had much lowe
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encapsula ion e iciency, so highe quan i y o sample was equi ed o pe o m he analysis and
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he e o e i in e e ed in he g ow h o he ungus due o he dilu ion o he aga . The e o e, he
353
ba s named BBG in Figu e 6 co espond o he suspensions wi hou cen i uga ion.
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17
355
Figu e 6. G ow h a ea o B. cine ea o he cen i uged suspensions o es e a ol. Ligh g ay:
356
high-shea emulsi ica ion. Do s: high p essu e emulsi ica ion. Da k g ay: high p essu e and
357
empe a u e emulsi ica ion. *: suspensions wi hou cen i uga ion. Signi ican ly di e en
358
esul s a e conside ed o p<0.05.
359
360
The o mula ion o es e a ol wi h leci hin and wi h he mix u e o leci hin and BBG had no
361
e ec on ungal g ow h, al hough some inhibi ion was achie ed wi h BBG wi hou mixing (up o
362
23%). The g ea es g ow h educ ion co esponded o he suspensions o YBG, anging he
363
inhibi ion be ween 44 and 53%. Howe e , his e ec was almos unno iceable o he mix u e
364
o YBG wi h leci hin: no inhibi ion was obse ed by high p essu e emulsi ica ion, and up o 15%
365
o he o he me hods. Excep in his case, no signi ican di e ence was no iced be ween each
366
emulsi ica ion me hod ega dless o he encapsula ing ma e ial. Pu e es e a ol did no
367
p oduce any inhibi ion o ungal g ow h.
368
Acco ding o hese esul s, he p esence o β-glucans in he o mula ion enhances he ungicide
369
ac ion o es e a ol wi h espec o he o mula ion wi h leci hin. Va ona e al., 2013 s udied
370
he pene a ion o liposomes o med by leci hin in o cell memb anes o di e en bac e ia. In
371
ha wo k, liposomes showed abili y o c oss cell walls when hey a e cons i u ed by
372
phospholipid laye s, as happens in g am-nega i e bac e ia. Howe e , hey could no c oss in o
373
he cell when he e we e some o he ba ie s, as in he case o he g am-posi i e bac e ia. B.
374
0
1000
2000
3000
4000
5000
6000
Con ol Lec LecBBG BBG* LecYBG YBG
G ow h a ea (mm2)
18
cine ea has a wo-laye cell wall composed o chi in and β-glucans (Tenbe ge, 2007). I is possible
375
ha liposomes could no pene a e in o ha ba ie and deli e es e a ol inside, and hus he
376
o mula ions wi h leci hin had no an i ungal e ec . On he con a y, es e a ol is be e
377
abso bed wi hin he cell wall o he ungus when i is encapsula ed in β-glucans, especially YBG,
378
which con ains he same β-glucan ype as he ungal cell wall (Zhu e al., 2016), so he inhibi o y
379
e ec on ungal g ow h is bigge . Ne e heless, he inhibi o y e ec achie ed wi h he liquid
380
o mula ions was no compa able o he one ob ained wi h solid pa icles o he same p oduc s
381
epo ed in a p e ious wo k (Salgado e al., 2015). Some s udies epo ed an elici o e ec o β-
382
glucans on he p oduc ion o es e a ol by some plan s, like Vi is ini e a (Vuong, F anco, &
383
Zhang, 2014). Thus, i is possible ha when he o mula ions wi h β-glucans a e applied in i o,
384
he concen a ion o es e a ol inc eases, and his could cause a g ea e ungal g ow h
385
inhibi ion.
386
387
4. Conclusions
388
A liquid o mula ion o es e a ol was de eloped and es ed agains B. cine ea, by h ee
389
di e en emulsi ica ion me hods: high-shea , high-p essu e and high p essu e and empe a u e
390
emulsi ica ion. As encapsula ing ma e ials, BBG and YBG we e used, bo h alone and mixed wi h
391
leci hin. YBG showed good encapsula ing p ope ies ega ding encapsula ion e iciency (74-
392
84%), besides p o iding 50% inhibi ion o ungal g ow h, he bes o all he p oduc s es ed. On
393
he con a y, BBG we e no as good as encapsula ing ma e ial because hei chains en angle and
394
o m a ne ins ead o single d ople s, hus ha ing low encapsula ion e iciency (31-54%). Also
395
hey showed lowe an i ungal ac i i y (a ound 20% inhibi ion). The o mula ion wi h leci hin also
396
showed high encapsula ion e iciency and small pa icle size, al hough i did no inhibi ungal
397
g ow h. When mixing β-glucans wi h leci hin, a educ ion on pa icle size was obse ed,
398
al hough he mixing also induced he o ma ion o c ys als o es e a ol inside he encapsula ed
399
19
pa icles in compa ison wi h β-glucans alone. The e o e, i was concluded ha he o mula ion
400
o es e a ol wi h β-glucans imp o ed he ac ion agains B. cine ea, p obably h ough an
401
enhanced abso p ion o he ac i e compound by he ungus. Compa ing he emulsi ica ion
402
me hods de eloped in his wo k, he e we e no big di e ences be ween hem on encapsula ion
403
e iciency and pa icle size. Ne e heless, by high p essu e and empe a u e emulsi ica ion,
404
amo phous es e a ol was ob ained inside he pa icles, whe eas i o med c ys als wi h he
405
o he me hods. Despi e his, he e ec on he educ ion o ungal g ow h was simila by all he
406
emulsi ica ion me hods o each ma e ial, wi hou signi ican di e ences be ween hem. As β-
407
glucans appea as a p omising e ec i e encapsula ing ma e ial o imp o e he an i ungal ac i i y
408
agains B. cine ea, he o mula ions could be de eloped wi h o he polyphenols in u u e wo ks.
409
I would be also in e es ing o assess i hey a e also e ec i e agains o he ungal species.
410
411
412
Acknowledgmen s
413
Au ho s acknowledge he Eu opean p ojec SHYMAN FP7-NMP-2011-LARGE-280983 and p ojec
414
PIP 063/147181 om Fundación Gene al o he Uni e si y o Valladolid o inancial suppo . M.
415
Salgado hanks o Minis e io de Educación, Cul u a y Depo e o he Fo mación de P o eso ado
416
Uni e si a io g an . S. Rod íguez-Rojo acknowledges o Minis e io de Economía, Indus ia y
417
Compe i i idad and Uni e sidad de Valladolid o he Juan de la Cie a ellowship.
418
419
20
REFERENCES
420
Ad ian, M., & Jeande , P. (2012). E ec s o es e a ol on he ul as uc u e o Bo y is cine ea
421
conidia and biological signi icance in plan /pa hogen in e ac ions. Fi o e apia, 83(8),
422
1345-1350.
423
Ad ian, M., Jeande , P., Veneau, J., Wes on, L., & Bessis, R. (1997). Biological ac i i y o
424
es e a ol, a s ilbenic compound om g ape ines, agains Bo y is cine ea, he causal
425
agen o g ay mold. Jou nal o Chemical Ecology, 23(7), 1689-1702.
426
Almag o, L., Ga cía-Pé ez, P., Belchí-Na a o, S., Sánchez-Pujal e, P. J., & Ped eño, M. A. (2016).
427
New s a egies o he use o Linum usi a issimum cell ac o ies o he p oduc ion o
428
bioac i e compounds. Plan Physiology and Biochemis y, 99, 73-78.
429
Aziz, A., Poinsso , B., Dai e, X., Ad ian, M., Bézie , A., Lambe , B., . . . Pugin, A. (2003). Lamina in
430
elici s de ense esponses in g ape ine and induces p o ec ion agains Bo y is cine ea
431
and Plasmopa a i icola. Molecula Plan -Mic obe In e ac ions, 16(12), 1118-1128.
432
Bebbe , D. P., & Gu , S. J. (2015). C op-des oying ungal and oomyce e pa hogens challenge
433
ood secu i y. Fungal Gene ics and Biology, 74, 62-64.
434
Bu kus, Z., & Temelli, F. (2000). S abiliza ion o emulsions and oams using ba ley be a-glucan.
435
Food Resea ch In e na ional, 33(1), 27-33.
436
Caddeo, C., Nache , A., Vassallo, A., A men ano, M. F., Pons, R., Fe nàndez-Busque s, X., . . .
437
Manconi, M. (2016). E ec o que ce in and es e a ol co-inco po a ed in liposomes
438
agains in lamma o y/oxida i e esponse associa ed wi h skin cance . In e na ional
439
Jou nal o Pha maceu ics, 513(1–2), 153-163.
440
Cox, D. J., Yang, R. (2008). Glucan p epa a ions. Pa en Applica ion Publica ion (Vol.
441
20080108114). Uni ed S a es.
442
C uz Cab al, L., Fe nández Pin o, V., & Pa ia ca, A. (2013). Applica ion o plan de i ed
443
compounds o con ol ungal spoilage and myco oxin p oduc ion in oods. In e na ional
444
Jou nal o Food Mic obiology, 166(1), 1-14.
445
Chang, X., Heene, E., Qiao, F., & Nick, P. (2011). The phy oalexin es e a ol egula es he
446
ini ia ion o hype sensi i e cell dea h in Vi is cell. PLoS ONE, 6(10), e26405.
447
de Paz, E., Ma ín, Á., Es ella, A., Rod íguez-Rojo, S., Ma ias, A. A., Dua e, C. M. M., & Coce o,
448
M. J. (2012). Fo mula ion o β-ca o ene by p ecipi a ion om p essu ized e hyl ace a e-
449
on-wa e emulsions o applica ion as na u al colo an . Food Hyd ocolloids, 26(1), 17-
450
27.
451
de Paz, E., Ma ín, Á., Ma eos, E., & Coce o, M. J. (2013). P oduc ion o wa e -soluble β-ca o ene
452
micella o mula ions by no el emulsion echniques. Chemical Enginee ing and
453
P ocessing: P ocess In ensi ica ion, 74, 90-96.
454
Fe nández-Ma , M. I., Ma eos, R., Ga cía-Pa illa, M. C., Pue as, B., & Can os-Villa , E. (2012).
455
Bioac i e compounds in wine: es e a ol, hyd oxy y osol and mela onin: A e iew.
456
Food Chemis y, 130(4), 797-813.
457
Filip, V., Plocko á, M., Šmid kal, J., Špičko á, Z., Melzoch, K., & Schmid , Š. (2003). Res e a ol
458
and i s an ioxidan and an imic obial e ec i eness. Food Chemis y, 83(4), 585-593.
459
Ga cía, M. C., Al a o, M. C., Cale o, N., & Muñoz, J. (2014). In luence o polysaccha ides on he
460
heology and s abiliza ion o α-pinene emulsions. Ca bohyd a e Polyme s, 105, 177-183.
461
Ga o, M. A., Ippoli o, A., Linsala a, V., Casca ano, N. A., Nig o, F., Vanadia, S., & Di Vene e, D.
462
(2011). Ac i i y o ex ac s om wild edible he bs agains pos ha es ungal diseases o
463
ui and ege ables. Pos ha es Biology and Technology, 61(1), 72-82.
464
Gonçal es, V. S. S., Rod íguez-Rojo, S., De Paz, E., Ma o, C., Ma ín, Á., & Coce o, M. J. (2015).
465
P oduc ion o wa e soluble que ce in o mula ions by p essu ized e hyl ace a e-in-
466
wa e emulsion echnique using na u al o igin su ac an s. Food Hyd ocolloids, 51, 295-
467
304.
468
21
Huang, X., Dai, Y., Cai, J., Zhong, N., Xiao, H., McClemen s, D. J., & Hu, K. (2017). Res e a ol
469
encapsula ion in co e-shell biopolyme nanopa icles: Impac on an ioxidan and
470
an icance ac i i ies. Food Hyd ocolloids, 64, 157-165.
471
Huang, X., Kakuda, Y., & Cui, W. (2001). Hyd ocolloids in emulsions: pa icle size dis ibu ion and
472
in e acial ac i i y. Food Hyd ocolloids, 15(4–6), 533-542.
473
Jeon, Y. O., Lee, J.-S., & Lee, H. G. (2016). Imp o ing solubili y, s abili y, and cellula up ake o
474
es e a ol by nanoencapsula ion wi h chi osan and γ-poly (glu amic acid). Colloids and
475
Su aces B: Bioin e aces, 147, 224-233.
476
Jung, H., Hwang, I., Sung, W., Kang, H., Kang, B., Seu, Y., & Lee, D. (2005). Fungicidal e ec o
477
es e a ol on human in ec ious ungi. A chi es o Pha macal Resea ch, 28(5), 557-560.
478
Kon ogio gos, V., Biliade is, C. G., Kiosseoglou, V., & Doxas akis, G. (2004). S abili y and heology
479
o egg-yolk-s abilized concen a ed emulsions con aining ce eal β-glucans o a ying
480
molecula size. Food Hyd ocolloids, 18(6), 987-998.
481
Laza idou, A., K i ikopoulou, K., & Biliade is, C. G. (2015). Ba ley β-glucan c yogels as
482
encapsula ion ca ie s o p o eins: Impac o molecula size on he mo-mechanical and
483
elease p ope ies. Bioac i e Ca bohyd a es and Die a y Fib e, 6(2), 99-108.
484
Ma i, M., Be olini, P., & P a ella, G. C. (2003). Non-con en ional me hods o he con ol o
485
pos -ha es pea diseases. Jou nal o Applied Mic obiology, 94(5), 761-766.
486
Ma ins, N., Ba os, L., Hen iques, M., Sil a, S., & Fe ei a, I. C. F. R. (2015). Ac i i y o phenolic
487
compounds om plan o igin agains Candida species. Indus ial C ops and P oduc s, 74,
488
648-670.
489
Mikkelsen, M. S., Jespe sen, B. M., Mehlsen, A., Engelsen, S. B., & F økiæ , H. (2014). Ce eal β-
490
glucan immune modula ing ac i i y depends on he polyme ine s uc u e. Food
491
Resea ch In e na ional, 62, 829-836.
492
Panebianco, A., Cas ello, I., Ci ille i, G., Pe one, G., Epi ani, F., Fe a a, M., . . . Vi ale, A. (2015).
493
De ec ion o Bo y is cine ea ield isola es wi h mul iple ungicide esis ance om able
494
g ape in Sicily. C op P o ec ion, 77, 65-73.
495
Puja a, N., Jambh unka , S., Wong, K. Y., McGuckin, M., & Popa , A. (2017). Enhanced colloidal
496
s abili y, solubili y and apid dissolu ion o es e a ol by nanocomplexa ion wi h soy
497
p o ein isola e. Jou nal o Colloid and In e ace Science, 488, 303-308.
498
Rashed, K., Ći ić, A., Glamočlija, J., & Soko ić, M. (2014). An ibac e ial and an i ungal ac i i ies
499
o me hanol ex ac and phenolic compounds om Diospy os i giniana L. Indus ial
500
C ops and P oduc s, 59, 210-215.
501
Ri e a-Hoyos, C. M., Mo ales-Ál a ez, E. D., Pou ou-Piñales, R. A., Ped oza-Rod íguez, A. M.,
502
Rod Íguez-Vázquez, R., & Delgado-Boada, J. M. (2013). Fungal laccases. Fungal Biology
503
Re iews, 27(3–4), 67-82.
504
Salgado, M., Rod íguez-Rojo, S., Al es-San os, F. M., & Coce o, M. J. (2015). Encapsula ion o
505
es e a ol on leci hin and β-glucans o enhance i s ac ion agains Bo y is cine ea.
506
Jou nal o Food Enginee ing, 165, 13-21.
507
Sánchez-Bas a do, N., Rome o, A., & Alonso, E. (2017). Ex ac ion o a abinoxylans om whea
508
b an using hyd o he mal p ocesses assis ed by he e ogeneous ca alys s. Ca bohyd a e
509
Polyme s, 160, 143-152.
510
Shi, G., Rao, L., Yu, H., Xiang, H., Yang, H., & Ji, R. (2008). S abiliza ion and encapsula ion o
511
pho osensi i e es e a ol wi hin yeas cell. In e na ional Jou nal o Pha maceu ics,
512
349(1–2), 83-93.
513
Soylu, E. M., Ku , Ş., & Soylu, S. (2010). In i o and in i o an i ungal ac i i ies o he essen ial
514
oils o a ious plan s agains oma o g ey mould disease agen Bo y is cine ea.
515
In e na ional Jou nal o Food Mic obiology, 143(3), 183-189.
516
Spada o, D., & Gullino, M. L. (2004). S a e o he a and u u e p ospec s o he biological con ol
517
o pos ha es ui diseases. In e na ional Jou nal o Food Mic obiology, 91(2), 185-194.
518

22
S e ić, T., Be ić, T., Ša ikin, K., Soko ić, M., Gođe ac, D., Dimkić, I., & S anko ić, S. (2014).
519
An i ungal ac i i y o selec ed essen ial oils agains ungi isola ed om medicinal plan .
520
Indus ial C ops and P oduc s, 55, 116-122.
521
Tenbe ge, K. (2007). Mo phology and cellula o ganisa ion in Bo y is in e ac ions wi h plan s.
522
In Y. Elad, B. Williamson, P. Tudzynski & N. Delen (Eds.), Bo y is: Biology, Pa hology and
523
Con ol (pp. 67-84): Sp inge Ne he lands
524
Thammaki i, S., Suphan ha ika, M., Phaesuwan, T., & Ve duyn, C. (2004). P epa a ion o spen
525
b ewe 's yeas β-glucans o po en ial applica ions in he ood indus y. In e na ional
526
Jou nal o Food Science & Technology, 39(1), 21-29.
527
Timpe io, A. M., D’Alessand o, A., Fagioni, M., Mag o, P., & Zolla, L. (2012). P oduc ion o he
528
phy oalexins ans- es e a ol and del a- ini e in in wo economy- ele an g ape
529
cul i a s upon in ec ion wi h Bo y is cine ea in ield condi ions. Plan Physiology and
530
Biochemis y, 50, 65-71.
531
Tiwa i, B. K., Vald amidis, V. P., O’ Donnell, C. P., Mu hukuma appan, K., Bou ke, P., & Cullen, P.
532
J. (2009). Applica ion o na u al an imic obials o ood p ese a ion. Jou nal o
533
Ag icul u al and Food Chemis y, 57(14), 5987-6000.
534
Tsai, M.-J., Lu, I. J., Fu, Y.-S., Fang, Y.-P., Huang, Y.-B., & Wu, P.-C. (2016). Nanoca ie s enhance
535
he ansde mal bioa ailabili y o es e a ol: In- i o and in- i o s udy. Colloids and
536
Su aces B: Bioin e aces, 148, 650-656.
537
Va ona, S., Rod íguez-Rojo, S., Ma ín, A., Coce o, M. J., Se a, A. T., C espo, T., & Dua e, C. M.
538
M. (2013). An imic obial ac i i y o la andin essen ial oil o mula ions agains h ee
539
pa hogenic ood-bo ne bac e ia. Indus ial C ops and P oduc s, 42, 243-250.
540
Vuong, T. V., F anco, C., & Zhang, W. (2014). T ea men s a egies o high es e a ol induc ion
541
in Vi is ini e a L. cell suspension cul u e. Bio echnology Repo s, 1-2, 15-21.
542
Williamson, B., Tudzynski, B., Tudzynski, P., & Van Kan, J. A. L. (2007). Bo y is cine ea: he cause
543
o g ey mould disease. Molecula Plan Pa hology, 8(5), 561-580.
544
Wu, J., Deng, X., Tian, B., Wang, L., & Xie, B. (2008). In e ac ions be ween oa β-glucan and
545
calco luo cha ac e ized by spec oscopic me hod. Jou nal o Ag icul u al and Food
546
Chemis y, 56, 1131-1137.
547
Wu, Z., Ming, J., Gao, R., Wang, Y., Liang, Q., Yu, H., & Zhao, G. (2011). Cha ac e iza ion and
548
an ioxidan ac i i y o he complex o ea polyphenols and oa β-glucan. Jou nal o
549
Ag icul u al and Food Chemis y, 59(19), 10737-10746.
550
Xiong, S., Mel on, L. D., Eas eal, A. J., & Siew, D. (2006). S abili y and an ioxidan ac i i y o black
551
cu an an hocyanins in solu ion and encapsula ed in glucan gel. Jou nal o Ag icul u al
552
and Food Chemis y, 54(17), 6201-6208.
553
Zhu, F., Du, B., & Xu, B. (2016). A c i ical e iew on p oduc ion and indus ial applica ions o be a-
554
glucans. Food Hyd ocolloids, 52, 275-288.
555
556
557