Spruce galactoglucomannan-stabilized emulsions as essential fatty acid delivery systems for functionalized drinkable yogurt and oat-based beverage

Vol.:(0123456789) 1 3 Eu opean Food Resea ch and Technology h ps://doi.o g/10.1007/s00217-019-03273-5 ORIGINAL PAPER Sp uce galac oglucomannan‑s abilized emulsions asessen ial a y acid deli e y sys ems o  unc ionalized d inkable yogu andoa ‑based be e age FabioValoppi1,2 · NdegwaMaina1 · Ma jaAllén1· Robe aMiglioli1,3· Pe iO.Kilpeläinen4· Ki siS.Mikkonen1,2 Recei ed: 18 No embe 2018 / Re ised: 14 Ma ch 2019 / Accep ed: 24 Ma ch 2019 © The Au ho (s) 2019 Abs ac The ood indus y has a conside able demand o unc ional oods, such as emulsions as deli e y sys em o omega-6 and omega-3 a y acids. Such deli e y sys ems mus be s abilized, ideally wi h a compound ha ul ills he c i e ia o bo h unc ionali y and sus ainabili y. Sp uce galac oglucomannans (GGM) a e no el, wood-de i ed, na u al, alue-added, e sa ile, mul i-pu pose emulsi ie s ha can physically s abilize oil-in-wa e emulsions while simul aneously p o ec ing he oil phase agains oxida ion. In his s udy, we p esen o he i s ime he use o GGM-s abilized emulsions as complex mul icom- ponen deli e y sys ems o omega-6 and omega-3 a y acids, i.e., (1) cod li e oil in d inkable yogu and (2) oa oil in a glu en- ee egan be e age. The emulsions and he esul ing unc ionalized be e ages we e cha ac e ized in e ms o hei physical s abili ies. In addi ion, unc ionalized, d inkable yogu was cha ac e ized in e ms o he oxida i e s abili y o he oil. Resul s highligh ed ha oil d ople s s abilized wi h GGM we e s able du ing s o age, agains he mal ea men , upon addi ion in o he be e ages, and GGM p o ec ed cod li e oil agains oxida ion in d inkable yogu . The esul s epo ed he e highligh he as po en ial o he use o wood lignocellulose-de i ed, mul i- unc ional hyd ocolloids in mode n oods. Keywo ds Galac oglucomannan· Emulsion· D inkable yogu · Oa -based be e age· Func ional ood Abb e ia ions GGM Galac oglucomannan HFY High- a yogu LFY Low- a yogu OOE Oa oil emulsion OBB Oa -based be e age In oduc ion Func ional oods a e a class o oods u s ha has p o en heal h bene i s when consumed on a egula basis. They educe he isk o speci ic ch onic diseases and imp o e consume s’ physical o men al well-being [1, 2]. This class o oods has been ecognized as one o he mos inno a i e a eas o esea ch in he ood indus y [3]. Indeed, ea ing hab- i s ha e a g ea e ec on heal h, and consume s’ inc eased awa eness o his aspec , he inc eased cos o heal h ca e, and inc eased li e expec ancy ha e boos ed he demand o unc ional oods conside ably [2]. As a esul , se e al ood indus ies ha e launched new unc ional oods in he global ma ke in he las decade. The global ma ke o unc ional oods was alued $168 billion in 2013 and is expec ed o be wo h mo e han $300 billion in 2020 [4]. Basically, unc ional oods a e p oduced h ough he en ichmen o oods u wi h bioac i e and essen ial Elec onic supplemen a y ma e ial The online e sion o his a icle (h ps ://doi.o g/10.1007/s0021 7-019-03273 -5) con ains supplemen a y ma e ial, which is a ailable o au ho ized use s. * Ki si S. Mikkonen ki si.s.mikk[email p o ec ed] 1 Depa men o Food andNu i ion, Uni e si y o Helsinki, Agnes Sjöbe gin Ka u 2, P.O. Box66, FI-0014Helsinki, Finland 2 Helsinki Ins i u e o Sus ainabili y Science, Facul y o Ag icul u e andFo es y, Uni e si y o Helsinki, Helsinki, Finland 3 Depa men o Food, En i onmen al andNu i ional Sciences (DeFENS), Uni e si à degli S udi di Milano, Via G. Celo ia 2, 20133Milan, I aly 4 Na u al Resou ce Ins i u e Finland (LUKE), Tie o ie 2, 02150Espoo, Finland Eu opean Food Resea ch and Technology 1 3 molecules, such as p o eins, i amins, ca bohyd a es (i.e., die a y ibe ), and a y acids (i.e., omega-3 and omega-6 a y acids) [5] o adding p e- and p o-bio ics [6, 7]. Be e - ages a e consumed in la ge quan i ies h oughou he wo ld, and his has made hem he pe ec a ge o unc ionaliza- ion. Cu en ly, he be e age sec o is he mos ac i e sec o in he p oduc ion o unc ional oods, mainly due o hei con enience and hei p o iding he possibili y o easily mee ing consume s’ needs. Comme cial unc ional be e - ages include ene gy d inks, spo d inks, ui and ege a- ble be e ages, dai y-based be e ages, and non-dai y-based be e ages, such as oa -based be e ages [8, 9]. The ex en and na u e o he en ichmen a y depending on he ype o be e age. Fo example, spo d inks a e en iched mainly wi h mine als and i amins, whe eas dai y-based unc ional be e ages commonly a e en iched wi h p obio ics, mine als, a y acids, and bioac i e pep ides [8]. The deli e y o lipophilic compounds in a hyd ophilic ood ma ix equi es a deli e y and p o ec i e sys em ha can accommoda e he added molecules, p o ec hese mol- ecules when hey a e added in o he ood ma ix, and esis ex e nal s esses, i.e., echnological p ocesses, such as pas- eu iza ion, and he cha ac e is ics o he ood ma ix, such as low pH and high ionic s eng h [5]. To ob ain deli e y sys ems wi h unable cha ac e is ics, a ca e ul s uc u al design app oach mus be used, and i mus be coupled wi h echnological app oaches, such as emulsi ica ion [5, 10]. Among he echnologies ha po en ially could be used o p oduce a deli e y sys em, emulsi ica ion has a g ea ad an- age because i can be used o gene a e ex emely unable sys ems wi h an encapsula ion e iciency up o 100% [11]. Howe e , he p ope ies o he emulsi ie a e e y impo an since emulsions se e as he deli e y sys ems in unc ional oods. Indeed, emulsi ie s mus keep he emulsion s able when applied in he ood ma ix and unde di e en en i on- men al s esses [12]. As an addi ional poin , he deli e y o iable bioac i e molecules ha unde go oxida i e deg ada- ion o en equi es he use o an ioxidan s o main ain hei physiological e ec in he inal p oduc [10]. Recen ly, we iden i ied galac oglucomannan- ich (GGM- ich) ex ac s ob ained om indus ial so wood by-p oduc s as no el, na u al, alue-added, emulsi ying and s abilizing agen s [13]. GGM a e plan cell wall he e opolysaccha ides, i.e., hemicelluloses, wi h simila ca bohyd a e s uc u e as ha o known hyd ocolloids: gua gum galac omannan, locus bean gum galac omannan, and konjac glucoman- nan [14–16]. GGM consis s o a linea backbone composed o β-(1 → 4)-d-glucopy anosyl and pa ially ace yla ed β-(1 → 4)-d-mannopy anosyl uni s, b anched wi h α- (1 → 6)-d-galac opy anosyl side g oups [17]. GGM also con- ain wood-de i ed ex ac i es and phenolic co- a ele s [18, 19], which may p o ide addi ional unc ionali y o GGM [20]. GGM can be ob ained in high yield om abundan ly a ailable wood sawdus using sa e and en i onmen - iendly p essu ized ho wa e ex ac ion (PHWE) [21]. Thus, hey may be an in e es ing and sus ainable op ion o emulsion s abilize s. We showed ha GGM e icien ly s abilized emulsions agains coalescence [20, 22, 23] and p o ec ed he oil phase in emulsions agains lipid oxida ion [20, 24]. The p esence o phenolic esidues a ached o GGMs was p oposed o be he key elemen o he GGMs’ in e acial and an ioxidan p ope ies. Indeed, i was hypo hesized ha he s abilizing mechanism in ol es phenolic compounds ha deli e and ancho GGMs a he in e ace wi h oil d ople s and induce s e ic s abiliza ion [20]. GGM and lignin a e classi ied as die a y ibe s [25]. In addi ion o he s abilizing and an ioxidan p ope ies, GGM may also induce heal h-p omo ing e ec s, as e iewed by Pi känen e al. [25]. A e legisla i ely- equi ed sa e y e al- ua ions o no el oods [25], he cha ac e is ics o GGMs can make hem a unc ionalizing and unc ional ing edien , which is e y a ac i e in he ood sec o . GGM could be used as a mul i-pu pose ing edien o deli e and p o ec omega-6 and omega-3 a y acids and ob ain unc ional p oduc s. To de elop new deli e y/p o ec i e sys ems, hey mus be es ed in complex mul i-componen ood ma ices. In his s udy, we p esen wo applica ions in which he unc ionali y o GGM was exploi ed in d inkable yogu (as an example o a dai y-based be e age) and oa -based be e - ages (as an example o a glu en- ee, egan be e age). The aim o his s udy was o explo e he po en ial o he use o GGM-s abilized emulsions o deli e unc ional compo- nen s in complex ood ma ices, i.e., o ob ain unc ional oods. We ocused on he physical s abili y o he unc ional oods as well as he abili y o GGMs o p o ec sensi i e compounds (such as labile a y acids) agains oxida ion in a unc ional, d inkable yogu . In pa icula , sp uce GGMs we e used o p epa e emulsions ha we e ich in oils ha con ained omega-6 and omega-3 a y acids, e.g., cod li e oil and oa oil. The esul s highligh ed he as po en ial o he use o wood lignocellulose-de i ed mul i unc ional hyd ocolloids in mode n unc ional oods. Ma e ials andme hods Ma e ials Oa oil con aining osema y ex ac as an ioxidan ( a y acid composi ion in Table1), β-glucan- ich lou con aining 34–56% β-glucan [26–28], and oa p o ein- ich powde con- aining 73–87% p o eins [26, 27], we e kindly dona ed by Oy Ka l Faze Ab (Finland). 2.5 and 0.4% (w/w) dai y a d ink- able yogu s (Valio A + na u al yogu , Finland; a con en epo ed by he manu ac u e ) we e pu chased in a local ma - ke . Cod li e oil wi h α- ocophe yl ace a e and ocophe yl Eu opean Food Resea ch and Technology 1 3 ex ac as an ioxidan s (Mölle , O kla Heal h, No way) was pu chased om a local ma ke and he co esponding oil wi hou he addi ion o an ioxidan was kindly p o ided by O kla Heal h ( a y acid composi ion in Table1). Galac o- glucomannans (GGMs) we e ex ac ed om sp uce sawdus using a p essu ized ho wa e low- h ough ex ac o pilo plan [21]. The ex ac was hen sp ay-d ied and s o ed in pouches p o ec ed om ligh . Po assium so ba e was pu - chased om Fluka (Swi ze land). Sodium hyd oxide was pu chased om J.T. Bake (The Ne he lands). All sol en s used o ex ac ions we e pu chased om Sigma-Ald ich and Me ck (Ge many). In e nal s anda d nonadecanoic acid me hyl es e and GLC-68-D mix u e o me hyl es e s o a y acids we e pu chased om Nu-Check P ep, Inc. (USA). Sample p epa a ion Emulsions Concen a ed emulsion con aining cod li e oil A concen- a ed emulsion con aining cod li e oil was p epa ed using 50% (w/w) cod li e oil (wi h and wi hou α- ocophe yl ace a e and ocophe yl ex ac ), 40% (w/w) wa e , and 10% (w/w) GGMs. Fi s , he GGMs we e mixed wi h ap wa e a oom empe a u e and le o e nigh o ensu e maxi- mum dissolu ion. Then, oil was added o he GGM solu- ion, and an emulsion was ob ained by homogenizing he sys em using a T-18 basic Ul a-Tu ax high-speed homog- enize (IKA, Ge many) a 11,000 pm o 4min ollowed by 16,000 pm o 1min. The concen a ed emulsion was s o ed a 4°C and analyzed o e a pe iod o 29days. The emulsion was p epa ed only using high-speed homogeniza- ion due o i s high iscosi y. Oa oil emulsions We p epa ed ou di e en ypes o oa oil-con aining emulsions (OOE). To conside he dilu ion e ec due o he p epa a ion o an oa -based be e age (see below), i s , concen a ed emulsions we e p epa ed and hen dilu ed wi h ap wa e o he inal a ios o GGMs and oil, i.e., 0.2/1.5, 0.5/1, 0.5/2, and 0.8/1.5. The emulsions we e labelled as OOE0.2/1.5, OOE0.5/1, OOE0.5/2, OOE0.8/1.5, whe e he subsc ip s indica e he pe cen ages o GGM and oil in he emulsion. The con en o oil in OOE sam- ples was selec ed o be in he same ange o ha o com- me cially a ailable oa -based be e ages. The GGMs we e mixed wi h ap wa e a oom empe a u e and le o e nigh o ensu e hei maximum dissolu ion. Then, oil was added, and he mix u e was placed in an Ul a-Tu ax homoge- nize a 11,000 pm o 5min o ob ain a coa se emulsion. Fine emulsions we e ob ained by homogenizing he coa se emulsions wi h h ee passes a a p essu e o 800 ba using a Mic o luidize 110Y high-p essu e homogenize (Mic o- luidics, USA) con igu ed wi h 75 μm Y- ype F20Y and 200μm Z- ype H30Z chambe s in se ies. Po assium so ba e was added o he emulsions a a 0.1% (w/w) concen a ion o a oid mic obial spoilage. The oa oil emulsions we e s o ed a 22°C and analyzed o e a pe iod o 28days. Func ionalized be e ages D inkable yogu Func ionalized d inkable yogu was ob ained by adding he concen a ed cod li e oil emulsions (wi h and wi hou added ocophe yl ace a e and ocophe yl ex ac ) o he yogu ha had 2.5 and 0.4% (w/w) dai y a con en , wi h inal concen a ions o cod li e oil o 0.5 and 1% (w/w). The samples we e labeled as ollows: HFY0.5, HFY1, LFY0.5, and LFY1, whe e HFY and LFY mean high- a yogu (2.5%) and low- a yogu (0.4%), espec- i ely. The numbe indica es he concen a ion o cod li e oil in he unc ionalized, d inkable yogu . Concen a ed emulsions also we e p epa ed using cod li e oil wi hou α- ocophe yl ace a e and ocophe yl ex ac o s udy he s a- bili y o he oil wi h espec o oxida ion. The samples we e mixed wi h an Ul a-Tu ax homogenize a 11,000 pm o 1min o acili a e he inco po a ion o he emulsion in o he yogu ma ix and o ensu e ha he yogu had he desi ed low p ope ies o d inking. Also, o compa ison, d ink- able yogu s wi hou an added emulsion we e ea ed using he same p ocedu e. The samples we e labeled as HFY0 and LFY0, and hey we e s o ed a 4°C and analyzed o e a pe iod o 29days. Table 1 Fa y acid composi ion (% w/w) o oa oil and cod li e oil n.d. no de ec ed Fa y acid Oa oil Cod li e oil C14:0 0.18 4.8 C14:1 n.d. 0.12 C16:0 15 11 C16:1 0.19 10 C18:0 1.7 2.1 C18:1 (n-9) 39 18 C18:1 (n-7) 0.73 5.2 C18:2 (n-6) 40 2.5 C18:3 (n-3) 1.6 1.1 C20:0 0.79 14 C20:1 (n-9) 0.15 n.d. C20:2 (n-6) n.d. 0.34 C20:3 (n-3) n.d. 0.14 C20:4 (n-6) n.d. 0.34 C20:5 (n-3) n.d. 9.6 C22:0 0.01 6.3 C22:1 0.18 0.74 C24:0 n.d. 1.3 C22:6 (n-3) + C24:1 n.d. 12 Eu opean Food Resea ch and Technology 1 3 Oa ‑based be e age The oa -based be e ages we e ob ained by adding bo h aqueous β-glucan- ich lou and oa p o ein suspensions o he oa oil emulsion. A β-glucan- en iched suspension was ob ained by s i ing 3.3% (w/ ) β-glucan- ich lou in ap wa e o e nigh a oom empe - a u e (a ound 22°C). Then, he suspension was hea ed o 85°C and s i ed o 2h ollowed by s i ing o an addi- ional 2h a oom empe a u e. The oa p o ein suspension was ob ained using a modi ied e sion o he me hod p o- posed by Yang e al. [29]. In pa icula , a suspension ha con ained 7.5% (w/ ) o oa p o eins was s i ed o e nigh a oom empe a u e. Then, he pH o he suspension was adjus ed o 8 wi h 1M NaOH o imp o e p o ein solubili y, and he suspension was au ocla ed a 115°C o 12min. A e eaching 80°C, he suspension was wi hd awn om he au ocla e and cooled o oom empe a u e du ing mag- ne ic s i ing. Finally, he oa -based be e age was ob ained by mixing he h ee componen s o ob ain inal β-glucan and p o ein concen a ions o 1 and 3% (w/w), espec i ely. The mixing was done by a T-18 basic Ul a-Tu ax high-speed homogenize (IKA, Ge many) a 11,000 pm o 2 min. Fou di e en samples we e ob ained, and hey we e labeled as OBB0.2/1.5, OBB0.5/1, OBB0.5/2, and OBB0.8/1.5, whe e he numbe s in he subsc ip s e e o he pe cen age o GGMs and oil in he oa -based be e ages, simila o hose epo ed o he oa oil emulsions in pa ag aph 2.2.1.2. The con en o oil in OBB samples was selec ed o be in he same ange o ha o comme cially a ailable oa -based be e ages. Po assium so ba e was added o he samples a a 0.1% (w/w) concen a ion o a oid mic obial spoilage. The inal pH o OBB samples was 6.9. Selec ed samples we e pas eu ized a 80°C o 10min. Samples we e s o ed a oom empe a u e (a ound 22°C) and analyzed o e a pe iod o 28days. Analy ical de e mina ions Fa y acid composi ion The composi ion o a y acids o cod li e oil and oa oil was assessed by acid-ca alyzed es e i ica ion and anses- e i ica ion ollowed by gas ch oma og aphic (GC) quan- i ica ion. The es e i ica ion and anses e i ica ion we e pe o med using 1% sul u ic acid in me hanol as desc ibed by Ch is ie and Han [30], wi h some modi ica ions. A ound 50mg o oils we e i s dissol ed in oluene, added wi h 1% sul u ic acid in me hanol and incuba ed a 85°C o 60min. Es e i ied a y acids we e ex ac ed using hep ane a e addi ion o sa u a ed sodium chlo ide solu ion. The hep ane laye was collec ed and d ied o e anhyd ous sodium sul a e. The GC analysis was pe o med using he GC-FID equip- men and he me hod desc ibed by Yang e al. [31], wi h some modi ica ions. B ie ly, he a y acid me hyl es e s we e analyzed injec ing in a spli mode (1:15) 1µL o sample a 250°C in a GC-FID (Agilen 6890N, USA) equipped wi h a used silica capilla y column Omegawax™ l250 (30m × 0.25mm × 0.25μm, SUPELCO®, USA). Helium a 1.1mL/min was used as ca ie gas. The o en empe a u e was p og ammed o inc ease om 180°C wi h a 3°C/min o 220°C and inally inc eased wi h a 6°C/min o 250°C (wi h a 12-min inal hold). Me hyl es e s o a y acids we e iden- i ied using a GLC-68-D (Nu-Chek P ep. Inc., USA) com- me cial mix u e o me hyl es e o a y acids and quan i ied using nonadecanoic acid me hyl es e as in e nal s anda d. Size anddis ibu ion o  heoil d ople s The Sau e (D [3, 2]) and De B oucke e (D [4, 3]) mean diame e s and he d ople size dis ibu ion o emulsions we e de e mined using a Mas e size 3000 s a ic ligh sca e ing appa a us moun ed wi h a Hyd o EV dispe sion accesso y (Mal e n Ins umen s L d, UK). The ins umen was con- olled by he Mas e size .3.62 (Mal e n Ins umen s L d, UK) applica ion so wa e. Be o e conduc ing he analyses, he emulsions we e u ned upside down gen ly en imes. Then, he emulsions we e added di ec ly in o he dispe sion accesso y which allowed hem o be dilu ed o a oid mul- iple sca e ing e ec s. The e ac i e indexes o wa e and oil we e 1.33 and 1.47, espec i ely. The mean diame e s and size dis ibu ion o he oil d ople s we e calcula ed as an a e age o h ee measu emen s o each o he wo samplings pe o med on each emulsion. Mic oscopy All emulsions, plain and unc ional yogu s, and oa -based be e ages we e analyzed using an AxioScope A1 op ical mic oscope (Ca l Zeiss Inc., Ge many) connec ed o a Axi- ocam MRm digi al came a (Ca l Zeiss, Inc., Ge many). Be o e analysis, he samples we e mixed by u ning he con aine upside down en imes. One d op o sample was placed in he middle o a glass slide and a glass co e slip was cen e ed abo e he d op. The samples we e analyzed using bo h 40 × and 100 × objec i e. Images we e acqui ed using Axio ision .4.7.1.0 (Ca l Zeiss, Inc., Ge many) appli- ca ion so wa e. Flow cu es Flow cu es o unc ionalized d inkable yogu s, oa oil emul- sions, and oa -based be e ages we e ob ained using a Rheo- lab QC heome e (An on Paa GmbH, Ge many) con olled by Rheoplus .3.61 so wa e (An on Paa GmbH, Ge many). The expe imen s we e pe o med using a concen ic cylinde geome y CC27 (cylinde diame e : 26.66mm, cup diame e : 28.84mm), and he measu emen s we e conduc ed a 10°C o d inkable yogu and a 22°C o oa oil-con aining emulsions Eu opean Food Resea ch and Technology 1 3 and he oa -based be e age. An RC6-RCS empe a u e con ol- le uni (Lauda, Ge many) was connec ed wi h he heome e and used o con ol he empe a u e. Be o e he samples we e analyzed, hey we e mixed by u ning he con aine upside down en imes. Flow cu es we e ob ained by inc easing he shea a e s ep-wise om 2 o 200s−1. Sedimen a ion The s abili y agains he g a i a ional sepa a ion o oa -based be e ages (OBB0.2/1.5, OBB0.5/1, OBB0.5/2, and OBB0.8/1.5) was de e mined using he me hod p oposed by Mi hosseini e al. [32]. In pa icula , 10mL o he oa -based be e age was ans- e ed in o a 15-mL Falcon ube. Du ing s o age, he olume (mL) o sedimen was measu ed, and he s abili y index (SI) was compu ed as ollows: whe e VT is he o al olume (mL) o he sample, VS is he olume (mL) o he syne esis laye in he op o he Falcon ube, and SI is he mean pe cen age ± SE de i ed om he wo expe imen al eplica es. Seconda y oxida ion p oduc s The seconda y oxida ion p oduc s o plain and unc ionalized d inkable yogu s (HFY0, HFY0.5, HFY1, LFY0, LFY0.5, and LFY1) ha con ain cod li e oil wi hou α- ocophe yl ace a e and ocophe yl ex ac we e ex ac ed by solid phase mic oex ac ion (SPME) and analyzed wi h gas ch oma og- aphy coupled wi h mass spec ome y (GC–MS), using he same me hod desc ibed by Leh onen e al. [24]. Th ee epli- ca es consis ing o app oxima ely 2g o sample ha had been weighed in ambe glass ials, he me ically sealed, and s o ed a 4°C in he da k we e wi hd awn o analysis. B ie ly, he ola ile compounds we e adso bed on o a DVB/CAR/PDMS ibe (Supelco, USA), eleased in he GC–MS injec o a 250°C o 10min, and analyzed using an HP 6890 se ies GC coupled wi h an Agilen 5973 MS (Agilen Technologies Inc., USA). They we e sepa a ed using a SPB-624 capilla y column (Supelco, USA) and a empe a u e p og am om 40 o 200°C wi h helium a a low a e o 0.7mL/min wi h he MS unning in ull-scan mode. The compounds we e iden i ied based on he compa ison o hei e en ion imes and mass spec a wi h hose o he s anda ds. Da a analysis All de e mina ions we e exp essed as he mean ± s anda d e o (SE) o a leas wo measu emen s om wo eplica es (n ≥ 2 × 2), i no o he wise speci ied. Linea eg ession SI (%) = VT − VS V T × 100 analysis by leas -squa es eg ession was pe o med, and he goodness o i was e alua ed on he basis o he s a is ical pa ame e s o i ing (R2, p alue, and s anda d e o ) and he esidual analysis. S a is ical signi icance was conside ed o p < 0.05. Resul s anddiscussion Cod oil emulsions The i s example o p oduc unc ionaliza ion was on d ink- able yogu using an emulsion s abilized by GGM highly loaded wi h omega-6 and omega-3 ich oil. D inkable yogu is a ood p oduc ha is consumed wo ldwide, and, cu en ly, i is used as a a ge ood o unc ionaliza ion [8]. We will i s explo e he highly oil-loaded emulsion, which was p e- pa ed o a oid he dilu ion e ec when he emulsion was added o he yogu . Figu e1a shows he mac oscopic appea ance o GGM- s abilized emulsion con aining 50% (w/w) cod li e oil (he ea e called concen a ed emulsion). The sample appea ed as a b ownish dispe sion. Du ing he ex ac ion Fig. 1 a Mac oscopic image o he eshly p epa ed, concen a ed emulsion ha con ained 50% cod li e oil and 10% GGM, b i s el- e an op ical mic oscopy image, and c d ople size dis ibu ion o emulsion du ing s o age a 4°C Eu opean Food Resea ch and Technology 1 3 p ocess, lignin and o he ex ac i es we e eco e ed along wi h GGM [21]. Lignin is a mac omolecule ha is com- posed o polyme ized monolignols (phenolic compounds) [33], and i s colo anges om ligh b own o da k b own [34]. The p esence o lignin and o he ex ac i es p obably is esponsible o he b ownish colo o he concen a ed emulsion. F om a mic oscopic pe spec i e, he concen a ed emul- sions appea ed as a polydispe se sys em in which a la ge numbe o oil d ople s wi h di e en diame e s was obse ed (Fig.1b). A e 15days o s o age a 4°C, no di e ences in he mo phology o he oil d ople s we e obse ed (da a no shown). Then, he s abili y o he concen a ed emul- sion o e ime was con i med by he d ople size dis ibu ion (Fig.1c). A main peak a a ound 3μm wi h a shoulde a a lowe size was isible o all s o age imes. The concen a ed emulsion was physically s able o 15days o s o age a 4°C, exhibi ing a cons an D [3, 2] and D [4, 3] o 1.75 ± 0.05 and 2.74 ± 0.02, espec i ely. The s abili y can be a ibu ed o bo h GGMs’ s abilizing abili y and he high mass ac ion o oil in he concen a ed emulsion. Indeed, i is known ha a high mass ac ion o oil inc eases he iscosi y o emul- sions, which, in u n, e a ds he possible b eakdown mecha- nisms, he eby inc easing he s abili y o he emulsion [12]. Concen a ed emulsions can be used as ing edien s o add o dilu e o ob ain he inal p oduc . The ac ha concen a ed emulsions con ain high amoun s o oil allows hese emul- sions o be s o ed o p olonged pe iods o ime and be added a he desi ed ime. Also, since concen a ed emulsions a e ing edien s, hey educe he cos s ela ed o he s o age and anspo a ion o he inal p oduc [35, 36]. D inkable yogu The concen a ed emulsion was added o d inkable yogu wi h 2.5 o 0.4% (w/w) a con en o ob ain inal concen a- ions o 0.5 and 1% (w/w) o cod li e oil in he inal p od- uc (samples HFY0.5, HFY1, LFY0.5, LFY1). The addi ion o he emulsion al e ed he colo o he d inkable yogu s sligh ly, om whi e o sligh ly b own (Fig.2). I is known ha colo changes in ood p oduc s modi y he pe cep ion and accep ance o he p oduc s [37]. Howe e , consume s end o accep senso ial changes in ood p oduc s ha a e en iched o unc ionalized compa ed o he espec i e plain oods [38]. Figu e2 shows he mic og aphs o he plain (HFY0 and LFY0) and unc ionalized d inkable yogu s (HFY0.5, HFY1, LFY0.5, LFY1). Cod li e oil d ople s in HFY0.5, HFY1, LFY0.5, LFY1 we e dis ibu ed e enly in he sam- ples and inco po a ed in o he p o ein–yogu ma ix. No di e ences we e no ed among he samples, indica ing ha he p esence o milk a did no a ec he s abili y o he oil d ople s. Also, no mo phological di e ences we e no ed be ween he oil d ople s in he concen a ed emulsion and he oil d ople s dispe sed in he d inkable yogu s (c . Figs.1b and 2). The s abili y o oil d ople s in d inkable yogu can be explained by he educed/absen in e ac ions be ween GGMs and caseins and he iscosi y o he sys em. Yogu is o med upon acidi ica ion induced by e men a- ion, and a h ee-dimensional ne wo k o clus e s and chains o caseins is o med due o educed elec os a ic epulsion be ween he casein molecules [39]. The ze a po en ial o caseins is sligh ly nega i e (close o ze o) a pH 3.5, and i dec eases as he pH alue inc eases [40]. In acidic con- di ions (pH = 4.5), GGMs ha e shown a ze a po en ial o abou − 10mV [13]. Thus, when he concen a ed emulsion comes in con ac wi h he casein ne wo k o he yogu , he weak elec os a ic o ces ha ac be ween he GGMs and he Fig. 2 Mac oscopic images (le o igh ) o HFY1, HFY0.5, and HFY0 and op ical mic oscopy images o LFY0, LFY0.5, LFY1, HFY0, HFY0.5, and HFY1 Eu opean Food Resea ch and Technology 1 3 caseins pe mi he inco po a ion o he concen a ed emul- sion in o he yogu ’s p o ein ma ix. To unde s and he e ec o he addi ion o he emulsion on he mechanical p ope ies o he d inkable yogu s, he low cu es o HFY0, HFY1, LFY0, and LFY1 we e de e - mined (Fig.3). Bo h yogu s (LFY0 and HFY0) had pseudo- plas ic beha io (Fig.3). HFY0 had a low cu e ha shi ed o lowe alues han ha o LFY0. This shi was due o he supp ession e ec ha a has on he elas ic componen o he yogu ’s p o ein ne wo k [41]. Howe e , e en hough HFY0 and LFY0 showed di e en low beha io s (Fig.3), i was possible o inco po a e he concen a ed emulsion in bo h ypes o yogu s. I is impo an o emembe ha is- cosi y can ha e a signi ican e ec on he s abili y o he oil d ople s in he yogu ’s p o ein ma ix. E en hough a small amoun o emulsion was p esen in HFY1 and LFY1, a sligh modi ica ion o he en iched samples’ low cu es was obse ed (Fig.3). In pa icula , he cu es we e shi ed a highe shea s esses, indica ing an inc ease in he iscos- i y. The same shi was obse ed o HYF0.5 and LFY0.5, e en hough he magni ude o he shi was lowe (da a no shown). I is known ha oil d ople s a e esponsible o inc eases in iscosi y due o he augmen ed ic ion among he pa icles, while oil olume ac ion is esponsible o he magni ude o he inc ease [12]. Howe e , in his s udy he magni ude o he inc ease is negligible om a p ac ical pe spec i e due o people’s low sensi i i y in de ec ing small changes in iscosi y [42]. No changes in he low beha io we e obse ed du ing 15days o s o age a 4°C (c . Figu e3 wi h Fig. S1 in Suppo ing In o ma ion). The ola ile oxida ion p oduc s we e e alua ed o e an ex ended s o age pe iod o 29days a 4°C o e alua e he p o ec i e e ec o GGMs on he cod li e oil ha was mixed in o he d inkable yogu s. No seconda y oxida ion p oduc s we e de ec ed du ing s o age in any o he unc ion- alized yogu s (HFY0.5, HFY1, LFY0.5, and LFY1). Addi- ionally, no di e ences among he samples we e de ec ed in he ch oma og ams. Thus, GGMs we e able o p o ec cod li e oil agains oxida ion in a complex ma ix, such as yogu , as highligh ed by he absence o seconda y oxida ion p oduc s, i.e., aldehydes, alcohols, and ke ones, du ing s o - age. Seconda y oxida ion p oduc s po en ially can be oxic o people since hey a e abso bed eadily and anspo ed o issues whe e hey can eac wi h p o eins, phospholipids, and nucleic acids [43]. The an ioxidan p ope y o GGM is a ibu ed o he ee and bound phenolic compounds ha eac wi h oxygen, he eby p o ec ing unsa u a ed a y acid moie ies in iacylglyce ol molecules [20, 24]. Thus, i is possible o use GGM o deli e he cod oil in yogu ha is ich in omega-6 and omega-3 a y acids and a oid he o ma ion o po en ially oxic molecules and ancidi y in d inkable yogu du ing s o age. Oa oil emulsions In his second case s udy, we epo he use o emulsions con aining oa oil and s abilized by GGM as a way o ob ain- ing unc ional, oa -based be e ages. Plan -based, non-dai y be e ages a e a apidly g owing segmen o unc ional ood p oduc s [44]. Oa -based be e ages a e an example o glu- en- ee milk al e na i es o egans. Oa s a e a glu en- ee ce eal ha can be consumed sa ely by celiac pa ien s, and his ce eal possibly can con ibu e o imp o emen s in he quali y o hei li es [45]. Oa β-glucan, con ained in oa be e ages, has a e y high capaci y o educing choles e ol, pos p andial glucose, and insulin; i s capaci y is e en highe han ha o ba ley β-glucan [46, 47]. Based on he bene icial e ec o oa β-glucan, he oa -based be e age p esen ed he e was made om oa β-glucan- ich lou , oa p o eins, and an emulsion s abilized using GGMs ha con ained oa oil, which is ich in omega-6 a y acids (Table1). All o he emulsions ha con ained oa oil we e isually homogeneous wi h a iscosi y compa able o ha o wa e (a ound 1mPa·s a 22°C). Du ing s o age, a hin c eaming laye was obse ed isually in all o he emulsions excep OOE0.5/1. All o he emulsions, excep OOE0.5/2, exhibi ed good s abili y o e ime. Samples wi h a highe GGM- o- oil a io (Fig.4, OOE0.8/1.5 and OOE0.8/1.5) had a main peak a ound 0.1μm, ollowed by mino peaks a highe d ople sizes. Du ing s o age, he main peak emained s able, while he mino peaks shi ed sligh ly a highe alues, indica ing good s abili y agains coalescence. Emulsions wi h lowe GGM- o-oil a ios (Fig.4, OOE0.2/1.5 and OOE0.5/2) had a main peak a 0.3μm and o he peaks o shoulde s a sizes g ea e han 2μm. In bo h emulsions, he peak a he lowe size emained s able o he en i e s o age pe iod, while he peaks and shoulde s ha we e la ge mo ed o highe alues, Fig. 3 Flow cu es o eshly p epa ed samples HFY0, HFY1, LFY0, and LFY1: he analyses we e conduc ed a 10°C. The ba s ep esen s anda d e o Eu opean Food Resea ch and Technology 1 3 indica ing a possible agg ega ion o coalescence o he oil d ople s. This phenomenon was mo e e iden in OOE0.5/2 han in OOE0.2/1.5. The s abili y o he emulsions ollowed he o de : OOE0.8/1.5 = OOE0.8/1.5 > OOE0.2/1.5 > OOE0.5/2, he la e sample being he mos uns able one. We also assessed he s abili y o he emulsion by e al- ua ing he Sau e (D [3, 2]) and De B oucke e (D [4, 3]) mean diame e s; D [3, 2] emained cons an du ing s o - age o all o he emulsions, bu D [4, 3] ended o inc ease wi h di e en magni udes du ing s o age. In pa icula , D [4, 3] inc eased sligh ly o OOE0.8/1.5 and OOE0.5/1 (ini- ial: 0.25 ± 0.05, inal: 0.45 ± 0.05µm) and inc eased o OOE0.2/1.5 (ini ial: 0.88 ± 0.02, inal: 2.21 ± 0.10µm), while a mo e ma ked inc ease was obse ed o OOE0.5/2 (ini ial: 2.76 ± 0.01, inal: 9.39 ± 0.10µm). The inc ease o he diame e s o he oil d ople s and he p esence o loccula ed oil d ople s also we e e alua ed using op ical mic oscopy. As an example, Fig.5 (le ) shows he eshly p epa ed emulsion o OOE0.8/1.5 [0.8% (w/w) GGM and 1.5% (w/w) oil] and a e 28days o s o age a 22°C ( igh ). No e ha he eshly p epa ed emulsion had inely dis ibu ed oil d ople s. A e 28days, some bigge d ople s and agg ega es we e isible, suppo ing he d ople size dis ibu ion da a. Gene ally, emulsions wi h highe GGM concen a ions and lowe oil concen a ions we e mo e s able du ing s o - age. The OOE, which had a GGM- o-oil a io equal o o less han 0.25, (Fig.4), unde wen pa ial coalescence du - ing s o age, while OOE wi h GGM- o-oil a io o a ound 0.5, (Figs.4 and 5a, b) we e mo e s able. Thus, he c i ical GGM- o-oil a io o ou OOE was be ween 0.25 and 0.5, lowe han ha ob ained by ou p e ious indings, which was be ween 1 and 2 [23]. P e iously, we p epa ed emulsions using apeseed oil and GGMs, which we e p ecipi a ed using Fig. 4 D ople size dis ibu- ion o OOE0.8/1.5, OOE0.5/1, OOE0.2/1.5, OOE0.5/2 du ing s o age a 22°C. Subsc ip s in abb e ia ions o he samples ep esen he pe cen ages o GGM and oil Fig. 5 Op ical mic oscopy images o eshly p epa ed OOE0.8/1.5 [emulsion con ain- ing 0.8% (w/w) GGM and 1.5% (w/w) oil] (le ) and a e 28days o s o age a 22°C ( igh ) Eu opean Food Resea ch and Technology 1 3 e hanol. The p ecipi a ion o GGMs by e hanol emo es he e hanol-soluble, low mola mass GGM oligo/polysac- cha ides and ee phenolic esidues, which migh help he physical and oxida i e s abiliza ion o he emulsion [20], and apeseed oil has a di e en a y acid composi ion han ha in Table1, which migh ha e inc eased he c i ical GGM- o-oil a io as well. The lowe he c i ical GGM- o-oil a io, he highe he abili y o he su ac an o physically s abilize an emulsion. Thus, using non-pu i ied GGMs, mo e s able emulsions can be ob ained compa ed o using e hanol- p ecipi a ed GGMs [20]. GGM migh ha e s abilized he oil d ople s by s e ic hind ance [22] he eby educing he in e ac ion be ween he oil d ople s and o he pa icles in he oa -based be e age. The mos s able emulsions (OOE0.8/1.5, OOE0.5/1) we e pas eu ized a 80°C o 10min o e alua e hei s abili y agains he mal ea men s. Bo h samples showed a d ople size dis ibu ion (Fig. S2, Suppo ing In o ma ion) and a mean d ople diame e compa able wi h hose o he non- pas eu ized samples (D [4, 3] o 0.25 ± 0.05 and D [3, 2] o 0.05 ± 0.01 o bo h pas eu ized and non-pas eu ized sam- ples), indica ing excellen s abili y o he emulsions when subjec ed o he pas eu iza ion ea men . Ou indings a e in ag eemen wi h he beha io highligh ed by Chanamai and McClemen s [48] on gum A abic and modi ied s a ch- s abilized emulsions. Indeed, polysaccha ides a e no p one o un olding o in e ace deso p ion induced by hea as in he case o p o ein-s abilized emulsions, so hey con e he mal s abili y o polysaccha ide-s abilized emulsions [12, 48]. Oa ‑based be e ages The OOE emulsions we e used o p oduce oa -based be - e ages by adding he oa p o ein and he β-glucan suspen- sions. Figu e6a shows an example o he esul ing be e - age (OBB0.5/1). The OBB be e ages we e b ownish in colo (Fig.6a). The colo o he OBB samples could ha e been due o OOE (in his case, he lignin compounds ha a e p esen in GGMs, as discussed p e iously) and β-glucan- ich lou , hus he colo coming om GGM is no expec ed o a ec he p oduc ’s accep abili y o consume s since he oa -based ing edien s ha e a simila colo . The samples appea ed as suspensions o pa icles ha ended o sedimen o e ime (Fig.6a, b). I is in e es ing o no e ha he s abili y o he oa -based be e ages in e ms o s abili y index (SI) seemed o be dependen on he GGM con en a he he GGM- o- oil a io (Fig.6b). The sedimen a ion was dependen on he emulsion o mula ion (Fig.6b). OBB0.2/1.5 had he highes s abili y index (SI) o e ime, whe eas, he sample based on he emulsion ha con ained he same amoun o oil (1.5%) bu mo e GGMs (0.8%), i.e., OBB0.8/1.5, had he lowes SI. The sedimen a ion could ha e been due o he p esence o insoluble pa icles in he be e age and o he agg ega ion o oa p o eins and oa β-glucan o e ime ha inc eased he dimensions o he pa icles, which a o ed phase sepa a ion [49, 50]. The p esence o oa p o eins and β-glucan led o he o ma ion o agg ega es ha inc eased o e ime, o ming a compac ma ix ha led o syne esis. Howe e , he agg e- ga es we e dispe sed easily by u ning he con aine upside down, again o ming a homogeneous dispe sion simila o ha obse ed in he eshly p epa ed samples. The educ ion o he SI also was e lec ed in he heo- logical p ope ies o he OBB samples, wi h all OBB sam- ples exhibi ing a pseudoplas ic beha io . O e ime, he samples ended o shi owa ds a mo e liquid-like beha - io (da a no shown). This ansi ion was accompanied by a ma ked educ ion o he iscosi y (Table2), which possibly was due o he agg ega ion o he suspended pa - icles which inco po a ed he oil d ople s, ollowed by syne esis. Indeed, when a sys em unde goes agg ega ion, he heological beha io o he esul ing ma e ial can be al e ed and esul in a educ ion o he iscosi y o he sys em i sel [12]. Howe e , oil did no sepa a e om he be e age; a he , i emained dispe sed as small oil d ople s which can be e-dispe sed by mixing he sample. No isible oil was de ec ed on op o he sample, which Fig. 6 Visual appea ance o OBB0.5/1 (a) and s abil- i y indexes (SI) o oa -based be e ages (b) a e 1, 7, and 28days o s o age a 22°C