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Protein-stabilized emulsions containing beta-carotene produced by premixmembrane emulsification

A. Trentin, S. De Lamo, C. Güell, F. López, M. Ferrando ⇑Departament d’Enginyeria Química, Universitat Rovira i Virgili, Av. Països Catalans 26, 43007 Tarragona, Spain

a r t i c l e i n f o

Article history:Received 29 November 2010Received in revised form 10 March 2011Accepted 12 March 2011Available online 24 March 2011

Keywords:Membrane emulsificationBeta-caroteneBSAWhey proteinFoulingO/W emulsions

a b s t r a c t

Protein-stabilized O/W emulsions containing beta-carotene were produced by premix membrane emul-sification (ME) using polymeric microfiltration membranes. Bovine serum albumin (BSA) and a whey pro-tein concentrate (WPC) were used as protein emulsifiers while a nonionic small-molecule surfactant,Tween 20, was used both as a control and co-emulsifier. Membrane fouling caused by WPC reduced moresignificantly transmembrane flux than that by BSA. Mixtures of WPC or BSA with Tween 20 reduced pro-tein membrane fouling and, simultaneously, decreased the mean droplet size. WPC/Tween 20 mixturesenable to produce emulsions with low polydispersion (span < 1) but with a significant membrane foulingwhile BSA/Tween 20 mixtures led to higher transmembrane fluxes although polydisperse emulsions(span = 7). During storage at 22 and 35 �C, the chemical degradation rate of emulsions with WPC/Tween20 was slower than those with BSA/Tween 20 whereas Tween 20-stabilized emulsions led to the highestrate of beta-carotene reduction during storage at 35 �C.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Delivery systems to encapsulate bioactive lipophilic com-pounds, such as oil-in-water (O/W) emulsions, have been reportedbecause of their interest to the food industry (McClements et al.,2009). These water-dispersible systems not only enable high-watercontent foods to be enriched with lipophilic carotenes but also toprevent them from oxidative deterioration. The physical and oxi-dative stability of oil-in-water emulsions strongly depends, amongother factors, on the type and concentration of the emulsifiersused. Polymeric amphiphilic molecules, such as food-grade pro-teins, are known to improve the interfacial coverage during emul-sification and so insure better encapsulation and controlled releaseof the bioactive compounds entrapped into the emulsion droplets(McClements, 2004). Whey proteins, bovine serum albumin(BSA), caseins, beta-lactoglobulin and other proteins have beenfound to increase the stability of oil-in-water emulsions (Dickinsonand Hong, 1994; Britten and Giroux, 1993; Al-Malah et al., 2000).Besides physical stability, another main concern regarding qualitydeterioration in oil-in-water emulsions is the susceptibility of lip-ids to oxidation. Proteins and peptides, in the form of emulsifiers oras ingredients added to the aqueous phase, have been reported toenhance the oxidative stability of O/W emulsions. Soy protein iso-late, sodium caseinate, whey protein isolate and BSA used as emul-sifiers can enhance the oxidative stability of oil-in-water emulsions

(Hu et al., 2003; Faraji et al., 2004; Velasco et al., 2004; Bonoli-Carbognin et al., 2008).

Although conventional methods for preparing emulsions usu-ally rely on stirring equipment, colloid mills, homogenizers, ultra-sonics or microfluidizers, over the last 20 years, there has been agrowing interest in membrane emulsification (ME). ME is highlyattractive because it is simple, requires potentially lower energydemands, needs less surfactant and results in narrow droplet sizedistributions (Charcosset, 2009; Van der Graaf et al., 2005). TheME process can be performed in different ways: direct ME and pre-mix ME. In the former, the dispersed phase is forced, using lowpressure, to permeate through a membrane into the continuousphase. The distinguishing feature of direct ME is that the resultingdroplet size is controlled primarily by the membrane and not bythe generation of turbulent droplet break-up. In Premix ME, a pre-liminarily emulsified coarse emulsion is forced through the mem-brane. This is achieved in a two-step process: (i) the twoimmiscible liquids are mixed together using a conventional stirrermixer to form a preliminary emulsion, which is then (ii) passedthrough the membrane. Premix ME has some advantages over di-rect ME: the optimal transmembrane fluxes with regard to dropletsize uniformity are one or two orders of magnitude higher thanthose of direct ME, and the experimental set-up is generally sim-pler and easier to control and operate than in direct ME (Naziret al., 2010; Vladisavljevic et al., 2004).

Emulsions and nanoemulsions containing carotenoids havemainly been produced by high pressure homogenization processessuch as microfluidization (Ax et al., 2003; Mao et al., 2009; Yin

0260-8774/$ - see front matter � 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.jfoodeng.2011.03.013

⇑ Corresponding author. Tel.: +34 977 558 551; fax: +34 977 559 621.E-mail address: montse.ferrando@urv.cat (M. Ferrando).

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et al., 2008; Kanafusa et al., 2007; Ribeiro et al., 2003, 2006). Premixmembrane emulsification with a polyamide microfiltration mem-brane was used by Ribeiro et al. (2005) to produce emulsions con-taining astaxanthin, while more recently Neves et al. (2008)reported using microchannel emulsification to encapsulate lipo-philic bioactive molecules such as beta-carotene. To stabilize theseemulsions research has been carried out into various emulsifierssuch as the mono-, di- and polyglycerol esters of fatty acids (Ribeiroet al., 2003; Yin et al., 2008), sucrose monolaurate (Neves et al., 2008;Ribeiro et al., 2006), Tween 20 (Mao et al., 2009; Ribeiro et al., 2005,2006), whey protein isolate (Mao et al., 2009; Ribeiro et al., 2005,2006), hydrolyzed whey protein isolate (Ribeiro et al., 2006) and so-dium caseinate (Kanafusa et al., 2007).

Trentin et al. (2009, 2010) reported using several microfiltrationpolymeric membranes in premix ME to produce protein-stabilizedoil-in-water emulsions. They found that when BSA was used as anemulsifier in premix ME during the production of O/W emulsions,it caused membrane fouling which reduced the transmembraneflux but which did not affect the droplet size diameter of theresulting emulsion.

The aim of this study was to carry out premix membrane emul-sification using polymeric microfiltration membranes in order toproduce protein-stabilized O/W emulsions containing beta-caro-tene. To determine to what extent proteins protected against theoxidative degradation of beta-carotene loaded in O/W emulsions,BSA and a whey protein concentrate were used as protein emulsi-fiers while a nonionic small-molecule surfactant, such as Tween20, was used both as a control and as a co-emulsifier. During pre-mix ME, the extent of membrane fouling caused by proteins wasdetermined in the nitrocellulose mixed esters (MCE) microfiltra-tion membranes. In addition, the physical and chemical stabilityof beta-carotene loaded O/W emulsions was monitored duringstorage at different temperatures to determine the protective ef-fect of proteins against oxidative deterioration.

2. Materials and methods

2.1. Materials

O/W emulsions with a 10% oil fraction (v/v) were preparedusing commercial sunflower oil as the dispersed phase, and dis-tilled, deionized water as the continuous phase. Beta-carotenepowder (Type I, synthetic, P93% (UV)) from Sigma–Aldrich wasdissolved in the sunflower oil. Tween 20 (polyoxyethylene sorbitanmonolaurate, from Sigma–Aldrich, Spain), BSA (albumin bovine/fraction V, from Acros Organics, Spain) and a whey protein concen-trate (WPC, Lactalbumin� 75 L, from Milei, Stuttgart, Germany)were used as emulsifiers and dissolved in the continuous phase.A phosphate buffer solution (PBS) was used to adjust the waterpH to 7 in emulsions that had been prepared using BSA or WPC.No adjustments were made to the water pH for emulsions pre-pared with Tween 20. Table 1 shows the specific formulation ofall the emulsions used in the present study.

The polymeric membrane discs that were used for premix MEwere 47 mm in diameter and made of nitrocellulose mixed ester

(MCE) (0.8 lm pore size, MWCT > 500 kDa, Sterlitech CorporationRef. MCEB0847100SG). The effective membrane diameter was41 mm, which gave an effective filtration area of 1.32 � 10�3 m2

for the membrane module.

2.2. Experimental set-up and emulsification procedure

O/W emulsions were prepared in a two step emulsificationsystem, as shown schematically in Fig. 1. The first step consistedof dissolving the beta-carotene in the sunflower oil and thenpreparing a coarse emulsion. To dissolve beta-carotene (meltingpoint of 180–182 �C) into the sunflower oil, 2/3 of oil volumewas heated at 160–180 �C while the rest, 1/3, was mixed withbeta-carotene at room temperature, forming a suspension. Whenoil reached the desired temperature it was added to the beta-carotene suspension and then mixed for 10 s to ensure completedissolution. The mixing time was well controlled to prevent thethermal degradation of the beta-carotene. The oil phase was thenadded to the water phase and both were mixed in a rotor–statorsystem (Ultra-Turrax�, model T18, IKA) at 15,500 rpm for 2 min,thus forming a coarse emulsion. In the second step, this emulsionwas loaded into the premix reservoir and forced with nitrogen(500 or 900 kPa) through the membrane. Table 1 shows the trans-membrane pressure used for each emulsion formulation. The fineemulsion resulting from this process was collected in an Erlen-meyer placed over a balance (BEL mark-3100). The balance wasconnected to a computer which recorded the time and mass everysecond. The premix procedure was repeated for 4–5 cycles in orderto obtain the final emulsion. Each experiment was repeated 3times and a new membrane was loaded into the membrane mod-ule at the beginning (1st cycle) of each experiment.

Permeate flux of the emulsion through the membrane duringpremix ME was calculated using mass and time data as follows:

J ¼ mt � A ð1Þ

where J (kg m�2 s�1) is the permeate flux, m (kg) is the total mass ofthe emulsion registered in the balance, t (s) is the time required forthe total mass to pass through the membrane and A (m2) is theeffective membrane area.

2.3. Droplet size distribution and interfacial tension

Samples of the O/W emulsions resulting from the rotor–statorand from the premix ME cycles were analyzed with a MalvernMastersizer 2000E to determine the droplet size distribution(mean droplet size and dispersion). Mean droplet size and disper-sion were expressed as the Sauter diameter (d32) and the relativespan factor, respectively, and they were measured at the end ofthe rotor stator emulsification and at the end of each of the 5 cy-cles. The relative span factor (from this point on, span) is a dimen-sionless parameter indicative of the uniformity of the droplet sizedistribution defined as:

Table 1Formulation of the O/W emulsions and transmembrane pressure used for each formulation.

Continuousphase

Dispersephase

Emulsifier pH Pressure(kPa)

MiliQ water(90% v/v)

Sunflower oil (10% v/v) containingdissolved beta-carotene (0.3% w/w)

Tween 20 (2% w/w) 6 500BSA (1% w/w) 7 900WPC (1% w/w) 7 900BSA (1% or 2% w/w) + Tween 20 (2% w/w) 7 500WPC (1% or 2% w/w) + Tween 20 (2% w/w) 7 500

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Span ¼ d90 � d10

d50ð2Þ

where d10, d50 and d90 are the representative diameters where 10%,50% and 90%, respectively, of the total emulsion volume are madeup of droplets with diameters smaller or equal to these values.The final emulsions were stored at three different temperatures:3, 22 and 35 �C and the change in droplet size distribution was fol-lowed for 21–36 days (depending on the emulsion stability).

The surface tension of the different emulsifier concentrationsand combinations was measured using the Drop Volume Method(ISO 9101:1987).

2.4. Beta-carotene extraction and analysis

To extract beta-carotene from the O/W emulsion, first 4 ml ofethanol (absolute, from J.T. Baker, Spain) and then 3 ml of hexane(purity >98%, from Sigma Aldrich, Spain) were added to 1 ml ofemulsion. The mixture was manually mixed and left to rest untila complete phase separation was achieved. The hexane phasewas taken out and another 3 ml of hexane were added to the eth-anol/emulsion solution until a colorless hexane was extracted,indicating that the b-carotene had been totally removed.

Total beta-carotene concentration was determined in the hexane/beta-carotene solution using spectrophotometry (CECIL CE20212000S series spectrophotometer) following the method describedby Britton et al. (1995). The results of the beta-carotene content wereexpressed as g/gemulsion.

To determine the chemical stability of the beta-carotene in thedifferent emulsions, the emulsions were stored, in darkness, atthree different temperatures: 4, 22 and 37 �C; and the beta-caro-tene concentration was monitored for 21–36 days (depending onthe emulsion stability to phase separation).

3. Results and discussion

3.1. Influence of emulsifiers on the transmembrane flux and dropletsize distribution during premix membrane emulsification

Fig. 2 shows the progress of d32 and transmembrane flux versusthe number of premix ME cycles obtained with MCE membranesand using a momoneric emulsifier (Tween 20) and two different

proteins (BSA and WPC) as single emulsifiers in the production ofbeta-carotene loaded O/W emulsions.

Results with the MCE membranes showed that transmembraneflux significantly decreased with the number of cycles regardless ofthe protein used as emulsifier, whereas this flux increased duringpremix ME of emulsions stabilized with Tween 20. In terms ofdroplet size, we observed the highest decrease during the 1st cycleof premix; that is, from the rotor–stator (cycle 0) to the first timethat the emulsion passed through the membrane, this being muchhigher for emulsions with WPC and Tween 20 than for those withBSA. No further droplet size reduction was observed with protein-stabilized O/W emulsions from the 2nd cycle on, although thosewith Tween 20 significantly reduced droplet size until the 3rd cy-cle. Emulsions stabilized with Tween 20 showed the smallest d32

after 4 cycles (1.6 ± 0.3 lm) followed by those obtained withWPC (3.3 ± 0.1 lm) and BSA (10.1 ± 1.1 lm). Similarly, emulsionswith Tween 20 showed the lowest dispersion, with span valuesof 0.82 whereas protein-stabilized emulsions showed higher spanvalues of 2.28 and 2.61 for BSA and WPC, respectively.

Protein fouling of the membrane, already detected during thepremix ME of BSA-stabilized O/W emulsions with MCE membranes(Trentin et al., 2010), would explain the anomalous transmem-brane flux decline observed during premix ME with BSA andWPC. Typically, when monomeric emulsifiers (e.g. Tween 20) in-stead of proteins are used, the transmembrane flux increases withthe number of cycles.

Vladisavljevic et al. (2004) considered that transmembranepressure applied during premix ME is used to overcome the fluxresistance and the droplet disruption resistance. According to theseauthors’ findings, transmembrane flux could be calculated directly,using Darcy’s law, from the ith cycle in which no further dropletsize reduction was observed. The equation for this process is:

Ji ¼DPtm

gporeðRm þ Rf ;iÞð3Þ

where gpore is the emulsion viscosity in the pores, Rm is the hydrau-lic resistance of a clean membrane, Rf,i is the overall fouling resis-tance for the ith cycle and Ji is the transmembrane flux for the ithcycle.

Trentin et al. (2010) used this approach to show how proteinfouling controlled the decrease in transmembrane flux during thepremix ME of emulsions that had been protein-stabilized with

160 –180 ºC

Sunflower oil

β-caroteneSunflower oil

MiliQ Water + emulsifier

Rotor-stator stage

Coarse Emulsion

N2

Membrane module

Fig. 1. Experimental set-up of the premix membrane emulsification process.

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BSA at different concentrations and with several organic microfil-tration membranes. In a similar way, the differences betweenBSA and WPC in terms of transmembrane flux (see Fig. 2) can beascribed to differing levels of protein membrane fouling with thefollowing equation:

JBSAi

JWPCi

¼gWPC

pore Rm þ RWPCf ;i

� �

gBSApore Rm þ RBSA

f ;i

� � � Ct ð4Þ

where Ct is a constant of value 5. Assuming that the emulsion vis-cosity in the pores was slightly different for the two emulsions,and that the transmembrane pressure was fully used to make theemulsion flow through the membrane (the droplet size was keptconstant for the 3 emulsifiers from the 3rd cycle, see Fig. 2), themuch higher overall fouling resistance can be attributed to WPCthan to BSA during the premix ME of beta-carotene loaded O/Wemulsions.

The surface-active properties of the emulsifier strongly deter-mine the droplet size distribution of the final emulsion becausethey play a double role of reducing the interfacial tension betweenoil and water and stabilizing the droplets against aggregation and/or coalescence. Given this, the interfacial tension at equilibriumwas determined for O/W emulsions with and without beta-caro-tene in sunflower oil for each of the 3 emulsifiers considered. Table2 shows how the presence of beta-carotene in the oil phase affectsthe equilibrium interfacial tension for each emulsifier differently.When adding beta-carotene (0.3%, w/w), BSA and Tween 20reduced interfacial tension by 13% and 18%, respectively, whereasWPC increased the interfacial tension by 37%. In these conditions,the interfacial tension between the oil with beta-carotene andthe water with BSA and WPC showed very close values of 4.95

and 5.20 Nm/m, respectively. Wackerbarth et al. (2009) showedthat the strong interactions between beta-carotene and proteins,particularly BSA, were able to form a stable protein–carotenoidcomplex whose surface–active properties were able to stabilizeO/W emulsions. The hydrophobic interactions between proteinand carotenoid, which are responsible for the complex formation,can explain the differences in surface-active properties (i.e. theinterfacial tensions) observed between emulsions with and with-out beta-carotene in the oil phase. Furthermore, the effect ofbeta-carotene on interfacial tension was different for each emulsi-fier because the molecular structures and conformations of BSA,WPC and Tween 20 may give rise to different distributions ofpolar/non polar groups and, thus, different interactions withbeta-carotene (McClements, 2004).

Considering only the values of interfacial tension shown inTable 2, BSA and WPC should lead to beta-carotene loaded emul-sions of a similar d32 when produced under the same process con-ditions (MCE membrane of 0.8 lm pore size and 900 kPa oftransmembrane pressure). In this case, however, after 4 cycles inemulsions with BSA the d32 was 3 times higher than that obtainedwith WPC even though the interfacial tension with BSA wasslightly lower (Fig. 2).

This can be explained by the fact that: (i) the values of equilib-rium interfacial tension shown in Table 2 do not provide informa-tion related to emulsifier adsorption kinetics to a freely formeddroplet and (ii) protein emulsifiers differ in the rate at which theyabsorb at the interface and in the minimum amount that isrequired to saturate the droplet surfaces (McClements, 2004).Although both BSA and WPC (mostly made of beta-lactoglobulin)are rigid globular proteins that take longer than small-molecularweight surfactants and other proteins to rearrange their structuresat the interface, of the two, WPC was able to stabilize smaller drop-lets than BSA (see Fig. 3).

Some authors have reported the use of emulsifier mixturesmade basically of a monomeric surfactant and a surface-activepolymer (typically a food-grade protein) to produce carotene-loaded O/W emulsions (Ribeiro et al., 2003, 2005; Mao et al.,2009). Nevertheless, limited information is available regardinghow these mixtures of emulsifiers affect the formation and stabil-ization of O/W emulsions containing carotenoids. Mao et al. (2009)found that when producing nanoemulsions enriched with beta-carotene, a mixture of Tween 20 with a whey protein isolate im-proved the stability of the beta-carotene nanoemulsion, especiallywhen the ratio of both emulsifiers was 1:1. Also, Trentin et al.(2010) showed that protein membrane fouling during premix MEcould be significantly reduced when using Tween 20/BSA mixturesinstead of single BSA to produce O/W emulsions.

MCE

Cycles0 1 2 3 4 5 6

Flux

(kg/

m2

s)

0

5

10

152% Tween 201% BSA 1% WPC

MEC

Cycles0 1 2 3 4 5 6

d 3.2 (

μ m)

0

2

4

6

8

10

12

14

162% Tween 20 (span 0.82)1% BSA (span 2.28) 1% WPC (span 2.61)

Fig. 2. Progress of transmembrane flux and droplet size (d32) with the number of cycles during premix ME with MCE microfiltration membranes and the emulsifiers Tween20, BSA and WPC. Error bars show standard deviation. Span values after the 5th cycle.

Table 2Interfacial tensions between sunflower oil and water at room temperature for eachemulsifier used.

Disperse phase Aqueous phase Interfacial tension(mN/m)

Sunflower oil with 0.3% of beta-carotene

Tween 20 (2%w/w)

2.34 ± 0.04

BSA (1% w/w) 4.95 ± 0.05WPC (1% w/w) 5.20 ± 0.05

Sunflower oil Tween 20 (2%w/w)

2.87 ± 0.04

BSA (1% w/w) 5.71 ± 0.07WPC (1% w/w) 3.26 ± 0.07

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In the light of this, Tween 20/protein (BSA or WPC) mixtureswere used as emulsifiers in order to simultaneously increase trans-membrane flux and decrease the emulsion droplet size and disper-sion during premix ME. Fig. 4 shows the progress of trasmembraneflux and d32 with the number of cycles during premix ME at 500 kPaof transmembrane pressure. Except for mixtures with 2% WPC,transmembrane flux showed a slight increase from cycle 3 (BSA1% and 2% and WPC 1%). Although the highest droplet size reductionwas in cycle 1, d32 significantly reduced until cycle 4 and from thispoint on it kept constant. After 5 cycles of premix ME with 1%BSA/2% Tween 20 and 2% BSA/2% Tween 20, d32 decreased to1.73 ± 0.19 and 1.76 ± 0.31 lm, respectively. Similarly, values ofd32 at cycle 5 were 1.28 ± 0.02 and 1.69 ± 0.49 lm for 1% WPC/Tween 20 and 2% WPC/Tween 20, respectively. When comparedwith experiments that used 2% Tween 20 as a single emulsifier(d32 = 1.19 ± 0.16 lm, cycle 5), we observed that none of the emul-sifier mixtures reduced droplets to the same extent as 2% Tween20, despite the higher overall content of emulsifier used in the mix-tures (3% and 4%). In all the cases, d32 was still above the mean mem-brane pore size (0.8 lm) that is typically considered to be theprocess limit for droplet size reduction in premix ME. The dropletsize dispersion of BSA/Tween 20-stabilized emulsions was muchhigher (span = 6.8–7.0) than that of emulsions with WPC/Tween20 (span = 0.7–1.0). When monitoring the droplet size histogramof these emulsions during premix ME (Fig. 5), we observed thatemulsions with 1% WPC/2% Tween 20 and 2% WPC/2% Tween 20showed monomodal distributions after 3 and 5 cycles, respectively.

Contrary, emulsions with BSA/Tween 20 mixtures led after eachemulsification cycle to bimodal distributions, even though the peakof bigger droplets (mean droplet size approx 10 lm) significantlyreduced after 5 cycles. Results on O/W emulsions stabilized with1% BSA/2% Tween 20 without beta-carotene dissolved but obtainedby premix ME under the same conditions, showed a different trend:the emulsions produced were monodisperse with span values of0.93 (Trentin et al., 2010). This points out how the presence ofbeta-carotene at the oil phase greatly influences the premix MEperformance.

Overall protein fouling resistance to the hydraulic membraneresistance ratio, Rf,i/Rm, was calculated for each emulsion mixturefrom Eq. (3) using the same assumptions as those considered inEq. (4) and assuming that overall fouling resistance of Tween 20is negligible (Vladisavljevic et al., 2004):

RTween 20=Proteinf ;i

Rm¼ JTween 20

i

JTween 20=Proteini

� 1 ð5Þ

Table 3 shows REmulsifier Mixturef ;i /Rm during the premix ME of beta-

carotene loaded O/W emulsions with BSA/Tween 20 and WPC/Tween 20. According to these results, the overall fouling resistancecaused 1–6% of the hydraulic resistance of the clean membrane forBSA/Tween 20 mixtures. Contrary WPC/Tween 20 mixtures led tomuch higher values of REmulsifier Mixture

f /Rm, causing much moreextended membrane fouling, particularly with 2% of WPC in theemulsifier mixture (REmulsifier Mixture

f ;i /Rm = 5.6).

Fig. 3. Droplet size histograms of O/W emulsions prepared with 1% BSA and 1% WPC after several premix ME cycles (n).

MCE

Cycles0 1 2 3 4 5 6

Flux

(kg/

m2

s)

0

5

10

15

202% Tween 20 1% BSA2% Tween 20 2% BSA 2% Tween 20 1% WPC2% Tween 20 2% WPC

MCE

Cycles0 1 2 3 4 5 6

d 32 (μ

m)

0

2

4

6

8

10

12

14

16

2% Tween 20 1% BSA (span 7.0)2% Tween 20 2% BSA (span 6.8)2% Tween 20 1% WPC (span 0.7)2% Tween 20 2% WPC (span 1.0)

Fig. 4. Transmembrane flux and droplet size (d32) versus the number of cycles during premix ME with a MCE membrane and different mixtures of the emulsifiers Tween 20,BSA and WPC. Span values after the 5th cycle.

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3.2. Chemical stability of beta-carotene loaded O/W emulsions duringstorage: effect of emulsifier on the kinetics of beta-carotenedegradation

The emulsifier formulation (Table 1) was selected on the basisof our previous experience of producing O/W emulsions with poly-meric membranes by premix ME. In particular, protein concentra-tion as single or co-emulsifier was used to prevent phaseseparation and complete membrane blockage during premix ME.For this reason, the total weight concentration of the emulsifierswas not kept constant in all the O/W emulsions investigated.

In the light of this, beta-carotene content and droplet size distri-bution of the emulsions were monitored over 35 days at 3, 22 and35 �C to determine the extent to which BSA and WPC used as singleemulsifiers or in mixtures with Tween 20 were able to increase thephysical and chemical stability of beta-carotene loaded O/W emul-sions produced by premix ME.

The premix ME process and the various formulations (Table 1)gave an average beta-carotene content of 0.026 ± 0.005 g/g emul-sion (C0). Fig. 6 depicts the ratio of beta-carotene content at anygiven moment to the initial content (C/C0), and the d32 of the

emulsions under these storage conditions. At a storage tempera-ture of 3 �C, beta-carotene content did not decrease significantlyover the whole storage period regardless of the emulsifier: onlyemulsions with 1% BSA showed a significant decrease after 21 days,resulting in a final reduction of 30%. These results correlate wellwith the progress of the d32 which remained constant for all theemulsions except those prepared with 1% BSA.

During 4 days of storage at 22 �C, non-significant reduction ofC/C0 was observed in emulsions with all of the single emulsifiers.After 4 days, 1% BSA led to the fastest kinetics of beta-carotenereduction, followed by 1% WPC. After 21 days, emulsions with 1%BSA showed the highest reduction in beta-carotene content(C/C0 = 0.36) together with an abrupt increase in mean droplet size(d32 = 28 lm), whereas emulsions with 1% WPC showed a signifi-cant but lower extent of beta-carotene reduction and droplet sizeincrease. In the case of emulsions with 2% Tween 20, C/C0 showeda smaller but significant decrease after 21 days (C/C0 = 0.87) andreduced to 0.71 after 35 storage days, even though d32 was con-stant throughout the whole period.

We observed an increase in emulsion stability with BSA/Tween20 mixtures stored at 22 �C compared to experiments with single1% BSA. Regarding the effect of protein concentration in the mix-tures, 1% BSA/Tween 20 led to slower kinetics of beta-carotenedegradation than 2% BSA/Tween 20. At these conditions, C/C0 re-mained above 0.9 for 14 and 4 storage days in emulsions with 1%BSA/Tween 20 and 2% BSA/Tween 20, respectively, while meandroplet size showed the same significant increase with both emul-sifier mixtures after 21 days’ storage.

Similarly, the chemical and physical stability of emulsions con-taining WPC/Tween 20 mixtures was better than that of emulsionswith 1% WPC. Although the WPC concentration did not show astrong effect on the decrease in beta-carotene content, we

Fig. 5. Droplet size histograms of beta-carotene loaded O/W emulsions prepared with BSA/Tween 20 and WPC/Tween 20 mixtures after several premix ME cycles (n).

Table 3The overall fouling resistance to the hydraulic membraneresistance ratio (REmulsifier Mixture

f /Rm) during premix ME of beta-carotene loaded O/W emulsions with BSA/Tween 20 and WPC/Tween 20.

Emulsifier mixture REmulsifier Mixturef /Rm

1% BSA + 2% Tween 20 0.062% BSA + 2% Tween 20 0.011% WPC + 2% Tween 20 0.622% WPC + 2% Tween 20 5.67

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observed that the reduction rate of beta-carotene content was sig-nificantly faster in emulsions with 1% WPC/Tween 20 than in thosewith 2% WPC/Tween 20. Storage of 7 and 14 days led to a C/C0

reduction of more than 0.9 for emulsions with 1% WPC/Tween 20and 2% WPC/Tween 20, respectively.

At 35 �C storage temperature, emulsions with 2% Tween 20showed the highest rate of beta-carotene reduction, with a com-plete degradation after 14 storage days, even though no significantchanges in d32 were observed over the whole period. Likewise,emulsions with a single protein were not chemically stable evenafter 4 days’ storage (C/C0 < 0.8), although the extent of beta-caro-tene degradation was lower than that in emulsions with 2% Tween20. Emulsions stabilized with protein/Tween 20 mixtures showedhigher stability than those stabilized with just a single protein asemulsifier; however, a significant degradation in beta-carotenewas observed after 7 days’ storage (0.91 < C/C0 < 0.84). In terms ofdroplet size, only emulsions with BSA/Tween 20 showed an

important increase in d32 after 14 storage days, whereas d32 didnot show significant changes in emulsions stabilized with WPC/Tween 20.

4. Conclusions

This study has shown that O/W emulsions with entrapped beta-carotene in the oil phase can be produced by applying a premix MEwith a polymeric membrane such as MCE and food grade proteinssuch as BSA and WPC as emulsifiers. Furthermore, the results dem-onstrate that the membrane fouling caused by the presence of pro-tein emulsifiers reduces transmembrane flux, with WPC causingsignificantly more membrane fouling than BSA.

The use of WPC and BSA combined with a nonionic surfactant,Tween 20, have been shown to reduce protein membrane foulingand, simultaneously, to decrease the mean droplet size to valuesclose to the operating limit, that is, the mean pore size (0.8 lm)

Storage Temperature = 3ºC

Time (day)0 10 20 30 40

C/C

0

0.0

0.2

0.4

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1.0

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2% Tween 20 1% BSA1% WPC2% Tween 20 1% BSA2% Tween 20 2% BSA2% Tween 20 1% WPC2% Tween 20 2% WPC

Storage Temperature = 3ºC

Time (day)0 10 20 30 40

d 32 (

μ m)

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Storage Temperature = 22ºC

Time (day)0 10 20 30 40

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1.2Storage Temperature = 22ºC

Time (day)0 10 20 30 40

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Storage Temperature = 35ºC

Time (day)

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Fig. 6. Progress of C/C0 and d32 of beta-carotene loaded O/W emulsions produced by premix ME with different emulsifiers (Tween 20, BSA and WPC) during storage at 3, 22and 35 �C.

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of the membrane. Even though WPC and BSA are globular proteinsand can be assumed, to some extent, to behave similarly at theinterface, their differences in molecular structure and conforma-tion seem to have a strong influence on the performance of premixME. While WPC/Tween 20 mixtures enable the production ofemulsions with low polydispersion (span < 1) and droplet sizesbetween 1.28 and 1.69 lm (albeit with a significant membranefouling), BSA/Tween 20 mixtures led to higher transmembranefluxes, although with higher polydisperse emulsions (span = 7). Itis also remarkable that the presence of beta-carotene at the oilphase led to much higher polydisperse emulsions than those ob-tained with the same formulation (10% oil fraction and 1% BSA/2% Tween 20) and under the same emulsification conditions butwithout beta-carotene.

Regarding the properties of O/W emulsions during storage, BSAand WPC as co-emulsifiers with Tween 20 increased the physicaland chemical stability compared to that of emulsions stabilizedwith single proteins. Furthermore, emulsions with WPC/Tween20 showed a slower rate of chemical degradation than those withBSA/Tween 20 during storage at 22 and 35 �C while Tween 20-stabilized emulsions led to the highest rate of beta-carotene reduc-tion during storage at 35 �C. Although a correlation was observedbetween chemical and physical emulsion stability, results obtainedat 35 �C of storage showed that chemical degradation of beta-carotene was not related to the physical destabilization of theemulsion alone. Accordingly, WPC and BSA demonstrated aprotecting effect against beta-carotene degradation during storageat 35 �C when compared to Tween 20.

Acknowledgment

The authors would like to acknowledge the Dirección Generalde Investigación, projects CTQ2004-01369/PPQ and CTQ2007-63002/PPQ, for providing financial support.

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