1021 Bulgrin Journl of griulturl Siene, 22 (No 6) 2016, 1021 1026 griulturl emy INFLUENCE OF WPC 80 DDITION ON COLOUR, DROPLET SIZE ND STORGE STBILITY OF W/O EMULSIONS M. TBSZEWSK 1* n D. NJGEBUER-LEJKO 2 1 University of griulture in Krkow, Deprtment of Fruit, Vegetle n Mushroom Proessing, 30-149 Krkow, Poln 2 University of griulture in Krkow, Deprtment of niml Prout Tehnology, 30-149 Krkow, Poln strt TBSZEWSK, M. n D. NJGEBUER-LEJKO, 2016. Influene of WPC 80 ition on olour, roplet size n storge stility of W/O emulsions. Bulg. J. gri. Si., 22: 1021 1026 Whey proteins re hrterize with ility to form n stilize foms n emulsions. The size n istriution of the isperse roplets signifintly ffet the stility n olour refletne ility (olour prmeters) of the emulsion. The W/O (wter-in-oil) emulsions with ifferent onentrtions of WPC 80 (whey protein onentrte with 80% protein), i.e., 0; 1.5; 5; 10; 15% were proue n nlyse. The emulsions iffere regring olour prmeters, the preprtions with higher protein supplementtions were rker, more re n yellow n their olour ws more sturte. The size of the isperse phse roplets ws not ffete up to 10% WPC 80 itive, wheres t higher protein onentrtions they were signifintly lrger. No signifint hnges of the roplet size in the isperse phse n of the qulity of e whey proteins were oserve throughout the experiment urtion. Key wors: whey protein onentrte, emulsions, SDS-PGE, roplet size Introution Emulsion is system onsisting of t lest two liqui immisile phses. Emulsions n e lssifie into two groups: simple emulsions n multiple emulsions. In simple emulsions, roplets of one liqui phse (isperse phse) re isperse in nother immisile liqui phse (ispersing phse). Simple emulsions re of two types: W/O (isperse phse wter; ontinuous phse oil) n O/W (isperse phse oil; ontinuous phse wter). Multiple emulsions re emulsions of emulsions, it mens tht isperse phse is t the sme times ontinuous phse for smller roplets of the liqui immisile phse (Pl, 2007, 2011). Emulsions re thermoynmilly unstle systems. They ten to e estilize with hnges oserve in their struture, size n istriution of the isperse roplets (Tholkov et l., 2006; Mnoi n Rizvi, 2009). The huge impt on the emulsion stility hs n ition of surfe tive gent. Surftnts hve two min funtions, one is to llow the formtion of emulsions through eresing the interfil tension etween phses n the seon is to stilize roplets in n emulsion (Pl, 2011). Milk protein, e.g., seins n whey proteins re nturl surftnts. Whey proteins (WPs) filitte the formtion n stiliztion of the emulsions. Protein sorption t the wter-oil interfe (t the surfe of the isperse roplets) of n W/O emulsion rete n interfil lyer whih exerts stiliztion effet through steri n eletrostti repulsion s WPs ontin hrge mino is (Sliwiński et l., 2003; Mnoi n Rizvi, 2009). The most importnt whey protein to stilize n emulsion is β ltogloulin (60% of the whey protein frtion), followe y α ltlumin (20%) n ovine serum lumin (3%) (Frrell et l., 2004; Drewiz n Dziu, 2005). *Corresponing uthor: m.tszewsk@ur.krkow.pl
1022 M. Tszewsk n D. Njgeuer-Lejko Low-egree hyrolyze WPs enhne the emulsion stility, wheres highly hyrolyze to short-hin pepties n free mino is my hve negtive impt on the proess of the emulsion formtion n further my erese the system stility (Ye n Singh, 2006). Emulsions prepre with the signifint inorportion of the hyrolyse WPs usully require ition of emulsifiers to ensure the preprtion stility (Ye et l., 2004). pperne, inluing olour, is etermine y omintion of ftors n is one of the most importnt prmeter of the prout qulity. Colour of emulsions minly epens on the sorption n sttering light ility. Light sorption in n emulsion epens on the onentrtion n type of hromophori mterils, e.g., yes, wheres light sttering is influene y the mirostruture, refrtive inex, size n onentrtion of ny prtile of the emulsion. The lightness of emulsions is influene y the onentrtion n size of the isperse roplets (Chnmi n MClements, 2001, 2002). Due to exellent physiohemil fetures, inluing emulsifying, foming n stilizing properties, WPs re wiely use s ingreients in foo (Ye n Singh, 2006) n osmeti (Klik et l., 2010) preprtions. However, these pplitions s well s sientifi reports re usully limite to the O/W emulsions. This work ws estlishe to estimte the effet of whey protein onentrte (WPC 80 v/v) ition on the roplet size of the isperse phse, emulsion olour prmeters n the ehviour of the e proteins uring storge in the reverse, i.e. W/O osmeti emulsion. Mterils n Methos Mterils Whey protein onentrte (WPC 80) ws purhse from Brtex (Psłęk, Poln). The oil phse omponents use in the stuy inlue: grpe (Vitis vinifer) see oil n olive (Ole europe) fruit oil otine from Olitli (Inex Foo, Luoń, Poln), lnolin (eps lne nhyrous), oo (Theorom o) utter, eeswx (Cer l) purhse from Phrm Cosmetis, (Krkow, Poln). The following itives were lso use: vitmin plmitte 1.0 MIU g -1 n l-α-toopheryl ette purhse from DSM (Mszzonów, Poln), soium enzote, itri i from Chempur (Piekry Śląskie, Poln), ethyl lohol from Polmos (Bielsko-Bił, Poln), lvener oil (Oleum lvnule) from Sn (Wrszw, Poln). ll other regents use were of nlytil gre. Preprtion of wter-in-oil (W/O) emulsions Wter-in-oil (W/O) emulsions with 0 (ontrol); 1.5%; 5%; 10%; 15% (w/w) ition of WPC 80 were proue y the reipe n proeures evelope t the University of griulture in Krkow (Tszewsk et l., 2012, 2014). The W/O emulsions were ompose of 60% of oil phse n 40% of wter phse (WPC 80 queous solution 39% n itives 1%) (w/w). Full esription of the preprtion proeure ws given in the previous work (Tszewsk et l., 2015). Prepre emulsions were store t mient temperture (20 ± 1ºC) n nlyse iretly fter proution s well s fter 5, 10 n 15 weeks of storge. Colour nlysis Mesurement of olour ws performe y the trnsmitne metho (mesuring geometry /0, illuminnt D65, rnge 400-700 nm t 20 nm intervls, optil pth length 20 mm) in terms of the CIEL system (1976) using the CM- 3500 spetrophotometer (Koni Minolt Sensing In., Jpn) s esrie y Wołosik (2009). The following CIE L*** prmeters were etermine: L* - lightness (from 0 (lk) to 100 (white)), * oorinte ((-) green (+) re olour), * oorinte ((-) lue (+) yellow olour), C hrom (mesure of olour intensity). Droplet size mesurement The roplet size (imeter) of the emulsions ws mesure using the MT5310L light mirosope (Meiji, Jpn) whih ws ouple with igitl mer (Motim 2300 3.0 megpixels, Germny) n PC with Moti Imge Plus 2.0 ML softwre (Germny). The metho esrie y Dyowsk (2006) with some moifitions ws pplie. The roplet size ws oserve in six fiels of view, using the 40 x mgnifitions. SDS-PGE nlysis nlyze emulsion tretments were mixe with SDS- PGE reuing smple uffer, triturte vigorously with porelin mortr n pestle until the wter phse ws expelle n left t mient temperture for 20 minutes so s to seprte phses. From the relese wter phse of the emulsion 20 μl of the smple ws tken followe y the ition of enturing uffer (40 μl). Eletrophoreti seprtion ws run using 5% thikening gel n 16% seprting gel with the use of Mini-PROTEN 3 ell pprtus (Bio-R Lortories, Rihmon, US). Seprte protein ns were stine with Coomssie rillint lue, then isoloure, snne n nlyze using GELSCN v. 2.0 softwre (Kuhrzyk, Poln). Sttistil nlysis ll types of emulsions were prepre n nlyse in three inepenent series. nlyses of olour were performe in uplite wheres imeter of the isperse rop-
Infl uene of Wp 80 ition on Colour, Droplet Size n Storge Stility of W/O Emulsions 1023 lets ws etermine in triplite. Results re expresse s men±stnr error. For the estimtion of the effet of WPC 80 ition n time of storge, the otine results were sujete to two-wy nlysis of vrine (NOV) n the signifine of ifferenes etween the mens ws etermine on the sis of Dunn test t the signifine level of P<0.05. The sttistil nlysis ws performe using Sttisti 10.0 softwre (SttSoft, In., Tuls, OK, US). Results n Disussion pperne, inluing olour, is n impression pereive y the sense of sight, whih usully etermines the will to possess or rejet ertin prout (Chntrpornhi et l., 1999; MClements, 2002). The nlyze emulsions iffere gretly s regrs olour profile Figure 1. Lightness of the emulsion (L*) notiely erese with the inrese of the WPC 80 onentrtion (P<0.05) with the exeption of the lk of signifint ifferene etween the emulsion without whey proteins n tht ontining 1.5% of WPC 80. This prmeter ws not sujete to ny hnges uring storge. The shre of green olour oorinte (-*) with the inresing WPC 80 itive erese signifintly in fvour of the re olour (+*). During storge of the emulsion without WPC 80 ition the signifint inrese of the green olour prmeter ws notie. The * olour oorinte in the emulsion fortifie with 1.5% of WPC 80 ws not sujete to ny hnges throughout the storge perio. In other whey protein tretments there ws oserve slow inrese of the re olour, n these hnges were sttistilly signifint. The shre of * olour ttriute s well s hrom inex (C) hnge signifintly (p<0.05) with the inrese of WPC 80 onentrtion, the shre of the yellow oorinte (+*) s well s olour intensity (C) of the emulsion inrese. On the other hn, there ws no signifint hnges in these olour prmeters uring storge. Evns et l. (2010) oserve tht the solutions ontining WPC 80 re not trnsprent n the L* vlue ereses with the reution of trnspreny. The higher WPC 80 ition the lower is trnspreny n L* vlue. This is onsistent with the results of the present stuy s with the inresing WPC fortifition the lightness of the emulsions erese. B B B C BC B B BC C e e e Fig. 1. The olour L***C prmeters of the emulsions with ifferent onentrtions of WPC 80 (%) uring storge. Storge t week: 0 (lnk olumns), 5 th (lk olumns), 10 th (grey olumns), 15 th (stripe olumns). Vertil rs orrespon to stnr error (n = 6), ifferent upper se/lower se letters enote sttistilly signifint ifferenes (P 0.05) etween the mens of the sme protein ontent uring storge or t the sme storge time for vrile pro tein ontents, respetively
1024 M. Tszewsk n D. Njgeuer-Lejko Fig. 2. The roplet sizes of emulsions with ifferent WPC 80 onentrtions uring room temperture storge. Vertil rs orrespon to stnr error (n=9), ifferent upper se/lower se letters enote sttistilly signifint ifferenes (P 0.05) etween the mens of the sme protein ontent uring storge or t the sme storge time for vrile protein ontents, respetively. The hnges of the olour profile ffete y the WPC 80 ition my result from the light refletne y the roplets of the isperse phse. MClements (2002) reporte, tht with inresing rius of the isperse phse roplets ove 0.1μm the vlue of L* olour prmeter ereses n t the sme time n inrese of * n * vlues is oserve. lso the reserh of Chntrpornhi n o-workers (1999) on the hnges of the L*, *, * olour prmeters s ffete y the roplet size of isperse phse in emulsions of ifferent olours inite the similr results for the re-oloure emulsion. Inrese of the roplet imeter of the internl phse up to 10 μm resulte in the very slight inrese of * n * vlues. On the other hn, lightness (L*) of the emulsion steily ereses with inresing roplet size of the internl phse. On the ontrry to emulsion lightness, the olour hrom (C) inreses with the inrese of the internl roplet rius ove 0.1 μm, s ws inite y the MClements (2002). The results of the present investigtions re only prtly onsistent with the ove sttements. In the our stuy the erese of the L* prmeter n onomitnt inrese of the *, * oorintes s well s olour sturtion (C) were oserve with inresing onentrtion of e WPC 80, however only the emulsion with 15% of WPC 80 ws hrterize y the signifintly higher roplet imeter thn other emulsions (~2 μm vs 1.5 μm). This inites tht the investigte W/O emulsions were muh more sensitive to the WPC 80 onentrtion-inue moifitions in olour prmeters thn to the hnges of the sizes of internl wter-phse roplets. However, it shoul e highlighte tht the isrepnies etween results my result from the emulsion type, s ite uthors investigte the hrteristis of reverse O/W emulsions. The size of the isperse phse roplets hs huge impt on the stility of preprtions s well s their olour profiles (Kiokis et l., 2007; MClements, 2002). The roplet imeter of the isperse phse in the emulsions ontining up to 10% of WPC 80 ws equl to pproximtely 1.5 μm n ws signifintly ifferent (P<0.05) from tht mesure for the emulsion with 15% WPC ition, whih mounte to ove 2μm (Figure 2 n 3). There were no sttistilly signifint hnges oserve of the roplet size uring storge, however slow teneny for lowering of the roplet size ws notie for the emulsions with 1.5 n 5% WPC 80 onentrtion. Similr results were otine y Dyowsk (2003), who reporte tht emulsion with 12% WPC ontent ws hrterize y the higher roplet imeter of the isperse phse (1-4 μm) thn i the emulsion with 6% itive of WPC. Reserh of Ye (2008) inites, tht the 2-10% ition of protein onentrte i not influene signifintly the roplet size of the ispersing phse. The sme ws otine in the present work for the emulsions with 5 n 10% ition of WPC 80. During proessing or storge proteolyti miroorgnisms, whih my penetrte into the prouts with rw m- Fig. 3. Photogrphs of the roplets in emulsions with ifferent onentrtions of WPC 80 (The numers in photos: 0, 1.5, 5, 10, 15 re the WPC 80 onentrtions (%)).
Infl uene of Wp 80 ition on Colour, Droplet Size n Storge Stility of W/O Emulsions 1025 knowlegements This Reserh ws finne y the Ministry of Siene n Higher Eution of the Repuli of Poln. Referenes Fig. 4. The SDS-PGE eletrophoretogrm of the whey proteins (WPC 80) e to the emulsions in the mount of (w/w) 1.5; 5; 10; 15% fter 15 weeks of storge terils, n e responsile for egrtion of the intt proteins into smller pepties (Sherze n Mushiolik, 2001) n thus they n lower the stility of the emulsion system. oring to the results of Euston n o-workers (2001) emulsions prepre with the ition of whey protein hyrolyste were less stle thn those prepre with whey protein onentrte. In the present stuy whey proteins e to the emulsion uring proution stge mintine their size within the 15-week storge perio. No egrtion of the polypeptie hin of the ertin proteins to the smller pepties ws oserve regrless of the pplie WPC 80 onentrtion (Figure 4). Conlusions ition of whey protein onentrte (WPC 80) into the wter-in-oil emulsion formul signifintly ffete the olour prmeters of the emulsion. s the WPC 80 ition inrese the emulsions eme rker n simultneously the inrese of the re n yellow oorintes n olour sturtion ws oserve. The imeter of the roplets of the isperse phse ws similr in the emulsions ontining WPC 80 in onentrtions up to 10%, wheres t higher onentrtion it inrese. No signifint hnges were oserve regring nlyse prmeters uring storge perio. Moreover, there ws not oserve ny egrtion of the whey proteins to the smller peptie frtion within the 15 weeks of the experiment. Chnmi, R. n D. J. MClements, 2001. Preition of emulsion olor from roplet hrteristis: ilute monoisperse oil-in-wter emulsions. Foo Hyroollois, 15: 83-91. Chntrpornhi, W., F. Clyesle n D. J. MClements, 1999. Influene of roplet hrteristis on the optil properties of olore oil-in-wter emulsions. Collois n Surfes : Physiohemil n Engineering spets, 155: 373-382. Drewiz, M. n J. Dziu, 2005. The struture of milk proteins versus their funtionl properties. Żywność. Nuk. Tehnologi. Jkość, 2 (43): 47-60 (Pl). Dyowsk, B. E., 2003. Moel whey protein onentrte-stilize O/W emulsions with inresing protein onentrtion. Milhwissenshft, 58: 170-173. Dyowsk, B. E., 2006. Nturl n moel O/W emulsions - omprison of physio-hemil properties. Milhwissenshft, 61: 249-252. Euston, S. R., S. R. Finnign n R. L. Hirst, 2001. Het-inue estiliztion of oil-in-wter emulsions forme from hyrolyze whey protein. Journl of griulturl n Foo Chemistry, 49: 5576-5583. Evns, J., J. Zulewsk, M. Newol, M.. Drke n D. M. Brno, 2010. Comprison of omposition n sensory properties of 80% whey protein n milk serum protein onentrtes. Journl of Diry Siene, 93: 1824-1843. Frrell, H. M., R. Jimenez-Flores, G. T. Blek, E. M. Brown, J. E. Butler, L. K. Cremer et l., 2004. Nomenltute of the proteins of ows milk-sixth revision. Journl of Diry Siene, 87: 1641-1674. Klik, D., D. Njgeuer-Lejko n T. Greg, 2010. Nonfoo pplitions of milk proteins review. In: M. Fnun (E.), Collois in Biotehnology. Chpter 7, CRC Press, Bo Rton, US, pp. 151-175. Kiokis, S., C. Dimkou n V. Oreopoulou, 2007. Effet of het tretment n roplet size on the oxitive stility of whey protein emulsions. Foo Chemistry, 105: 94-100. Mnoi, K. n S. S. H. Rizvi, 2009. Emulsifition mehnisms n hrteriztions of ol, gel-like emulsions proue from texturize whey protein onentrte. Foo Hyroollois, 23: 1837-1847. MClements, D. J., 2002. Colloil sis of emulsion olor. Current Opinion in Colloi n Interfe Siene, 7: 451-455. MClements, D. J., 2002. Theoretil preition of emulsion olor. vnes in Colloi n Interfe Siene, 97: 63-89. Pl, R., 2007. Rheology of oule emulsions. Journl of Colloi n Interfe Siene, 307: 509-515. Pl, R., 2011. Rheology of simple n multiple emulsions. Current Opinion in Colloi & Interfe Siene, 16: 41-60.
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