Journal of Food Engineering 113 (2012) 136–142

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Journal of Food Engineering

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Effect of homogenization conditions on properties of gelatin–olive oilcomposite films

Wen Ma a, Chuan-He Tang a, Shou-Wei Yin a,b,⇑, Xiao-Quan Yang a, Jun-Ru Qi a, Ning Xia c

a Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, PR Chinab State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, PR Chinac Department of Light Industry and Food Engineering, Guangxi University, Nanning 530004, PR China

a r t i c l e i n f o

Article history:Received 13 February 2012Received in revised form 6 May 2012Accepted 7 May 2012Available online 15 May 2012

Keywords:MicrofluidizationOlive oilParticle sizeMicrostructurePhysical propertiesFilms

0260-8774/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.jfoodeng.2012.05.007

⇑ Corresponding author at: Department of Food ScChina University of Technology, Guangzhou 510687114262; fax: +86 20 87114263.

E-mail address: feysw@scut.edu.cn (S.-W. Yin).

a b s t r a c t

Gelatin–olive oil composite films were prepared through emulsification to improve water barrier abilityof gelatin-based films. The effects of homogenization conditions of film-forming dispersions (FFD) onlipid droplets distributions in the FFD and films were evaluated and compared. Some selected physicalproperties, e.g., water vapor permeability (WVP), microstructure of the films were also evaluated. Therotor–stator homogenizer provided a lower energy input and so the largest particles were observed inthe related FFD and films. These films exhibited excellent water barrier ability, but poor mechanical resis-tance, extensibility and transparency. The microfluidizer provided the FFD with lower and narrower par-ticle size distributions, promoting mechanical resistance, extensibility and transparency of the films. Thephysical properties of the emulsified films were dependent on the special microfluidization pressure orcycle used, e.g., the WVP of the films decreased upon increasing microfluidization pressure or cycle.The present results indicated that the microfluidizer can be used to modulate lipid droplets in the FFD,thus films’ properties.

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1. Introduction

The large majority of the polymers presently utilized are of syn-thetic origin, their biocompatibility and biodegradability are extre-mely limited with respect to the biopolymers such as protein,polysaccharide and lipid. Biopolymer-based films have shownpromising potential as a substitute of petroleum-based plasticpackage films to reduce environmental impact. The characteristicsof biopolymer-based films are determined mainly by chemical nat-ure of the biopolymer. Proteins or polysaccharide-based films havegood mechanical behavior, but poor moisture barrier propertiesdue to their hydrophilic nature (Kristo et al., 2007). On the otherhand, although lipids yield films with good water vapor barrierproperties, they are brittle (Perez-Gago and Krochta, 2001). Amongthe biopolymer-based films, protein-based films are the mostattractive due to their nutritional value as well as impressive gasbarrier properties (Ou et al., 2004). Gelatin, a natural polymer fromanimal by-products through acid or alkaline hydrolysis, was pos-sessed of great potential to form edible/biodegradable films dueto its abundance, biodegradability, low cost (Bigi et al., 2002).However, the gelatin films prepared from bovine and/or porcine

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skins presented poor water vapor barrier ability (Sobral et al.,2001).

In order to obtain protein-lipid composite films with improvedwater barrier ability, a lipid can be incorporated into the proteinmatrix by laminate techniques to form bi-layer films, or dispersinga lipid in the protein aqueous solutions to obtain an emulsifiedfilm. Bi-layer films tend to delaminate over time, develop pinholesor cracks and exhibit non-uniform surface (Quezada Gallo et al.,2000). As concerns emulsified films, they require only one filmo-genic emulsion casting and one drying process, those films exhibitgood mechanical and adhesive properties. However, they are lessefficient water vapor barriers due to the fact that water vaporcan migrate through the continuous hydrophilic matrix (QuezadaGallo et al., 2000). Thus, small particles and a high emulsion stabil-ity during the film drying give rise to a homogeneous distributionof the lipid particles in the film, which in turn contributes to amore efficient control of water transfer (Debeaufort et al., 1993).

Various homogenization techniques, e.g., the rotor–statorhomogenization and microfluidization techniques, may be usedto modulate lipid droplets in the filmogenic dispersions. Rotor–stator systems are often used in the food industry to homogenizedifferent media and high viscosity immiscible liquids, reachingparticle sizes in the range of 1 lm (Urban et al., 2006). High pres-sure homogenizers with process pressures of up to 100 MPa arecommonly used to reduce the size of lipid particles, giving rise toparticles much smaller than 1 lm. Nowadays, novel homogenizers

W. Ma et al. / Journal of Food Engineering 113 (2012) 136–142 137

such as microfluidizers can produce pressures of 200 MPa or high-er, providing dispersions or emulsions with narrower particle dis-tributions due to the high shear stresses developed in themicrochannels of the interaction chamber (Sherwin et al., 1998;Strawbridge et al., 1995). The presence of a large number of uni-formly dispersed spherical particles in films may provoke an in-crease of the tortuosity factor for mass transfer in the continuousmatrix. However, there are few studies about the effects of varioushomogenization techniques, especially the microfluidizer on theproperties of film-forming dispersions as well as the related films.

Of vegetable oils, olive has received the most attention in meatproducts reformulation, chiefly as a source of MUFAs (mono unsat-urated fatty acids). Olive oil has a high biological value due to thepresence of a favorable mix of predominantly MUFAs and naturalantioxidants, and olive oil intake is associated with various positivehealth benefits in human health (López-Miranda et al., 2006). Thus,the objective of this work is to develop gelatin/olive oil emulsifiedfilms through emulsification technique to improve water barrierability of gelatin-based films. The effects of homogenization condi-tions of the FFD on lipid droplets size in filmogenic dispersions andemulsified films were evaluated and compared. Some selectedphysical properties, e.g., water vapor permeability (WVP), micro-structure of the films were also evaluated. In addition, the possiblerelationship between some selected physical properties and micro-structure (lipid droplets size) of emulsified films will also bediscussed.

2. Materials and methods

2.1. Materials

Bovine hide gelatin type B (Bloom 150), Glycerol (98% reagentgrade) was purchased from Sigma Chemical Co. (St. Louis, MO,USA). Original olive oil was purchased from a local supermarket(Guangzhou, China). All other chemicals used were of analyticalor better grade.

2.2. Preparation of film-forming emulsions

The film-forming dispersions (FFD) contained 5% (w/w) gelatinand glycerol was used as a plasticizer by using a glycerol-to-pro-tein ratio of 0.4:1 (w/w). The lipid-to-protein ratio in the emulsi-fied films was 0.2. So, the mass fraction of solid components inthe films was: 62.5% gelatin, 25% glycerol, and 12.5% lipid. ThepH of the solution was appropriately adjusted to 8 with 0.1 MNaOH. The FFD was heated (60 �C) to favor the oil dispersion inthe system. The emulsification process was performed by using arotor–stator homogenizer (Ultraturax T25, Janke & Kunkel, Ger-many) in a first step (at about 40 �C). Some FFD were homogenizedin a second step using a M-110EH microfluidizer (MicrofluidicsInternational Corp., Newton, Massachusetts, USA) as described in

Table 1Volume-surface mean diameter (D3,2) and weight mean diameter (D4,3) of gelatin/olive oi

Method First stepA Second stepB

A - -B 4 min 10000 rpm -C 4 min 10000 rpm 1 Cycle at 69 MD 4 min 10000 rpm 3 Cycles at 69E 4 min 10000 rpm 1 Cycle at 138F 4 min 10000 rpm 3 Cycles at 13

Superscript letters (a-e) indicate significant (P < 0.05) difference within the same columA The FFD was homogenized by rotor–stator homogenizer.B The FFD was homogenized by a M-110EH microfluidizer.C Values were expressed as means and standard deviations of triplicate measurement

Table 1. The temperature of the FFD increased to 50–60 �C aftermicrofluidization treatment, in a pressure or cycle dependent man-ner. The control samples were prepared using the same men-tioned-above procedure, except without addition of olive oil.

2.3. Particle size distribution of the film-forming dispersions

The particle size distribution of the FFD was measured by usinga laser diffractometer (MasterSizer 2000, Malvern Instruments,UK). The samples were diluted in de-ionized water at 2000 rpmuntil an obscuration rate of 10% was obtained. The Mie theorywas applied by considering the following optical properties for ol-ive oil droplets: a refractive index of 1.46. Each FFD was measuredin triplicate. Weight mean diameter (D4,3) and volume-surfacemean diameter (D3,2) were obtained according to eqs. (1) and (2).

D4;3 ¼P

nid4i

Pnid

3i

ð1Þ

D3;2 ¼P

nid3i

Pnid

2i

ð2Þ

ni is the number of droplets of a determined size range and di is thedroplet diameter.

2.4. Preparation of films

Following degassing under vacuum, the film-forming solutionwas cast onto rimmed, leveled glass plates coated with polyethyl-ene films (Clorox China Co. Ltd., Guangzhou, China). The film thick-ness was controlled by casting the same solids (2.8 g) on each plate(18 � 20 cm). The castings were air-dried in a thermostatic andhumidistatic chamber [25 ± 1 �C, 50 ± 5% relative humidity (RH)]for 48 h, then the films were peeled off the plates, and variousspecimens for physical property testing were cut. Specimens of2.5 � 8 cm rectangular strips were for tensile testing, and2 � 2 cm squares for moisture content (MC) and total soluble mass(TSM) measurements.

2.5. Moisture content (MC) and total soluble mass (TSM)

MC of films was determined by oven-drying at 105 ± 2 �C for24 h, and expressed as the percentage of initial film weight lostduring drying. TSM of each film was determined as the percentageof film dry matter solubilized after 24 h immersion in de-ionizedwater. Three randomly selected 2 � 2 cm samples from each typeof film were used to determine the TSM. The film specimens wereplaced in a 50 mL cuvette containing 30 mL of de-ionized water.The cuvettes were covered with polyethylene films (Clorox ChinaCo. Ltd., Guangzhou, China) and incubated in a shaking water bathat 25 �C for 24 h with gentle vibrating. Undissolved dry matter was

l film-forming dispersion (FFD) as affected by homogenization conditions.

D3,2C D4,3

C

- -3.090 ± 0.005a 13.99 ± 0.044a

Pa 0.570 ± 0.007c 0.871 ± 0.002d

MPa 0.396 ± 0.007e 0.931 ± 0.001c

MPa 0.433 ± 0.003d 0.667 ± 0.003e

8 MPa 1.621 ± 0.003b 3.680 ± 0.023b

n.

s.

138 W. Ma et al. / Journal of Food Engineering 113 (2012) 136–142

determined by removing the film pieces from the beakers, gentlyrinsing them with de-ionized water, and then oven-drying therinsed films (105 �C, 24 h).

2.6. Tensile strength (TS) and elongation at break (EAB)

TS and EAB were measured using a TA-XTplus texture analyzer(Stable Micro Systems, London, UK). Samples were preconditionedat 25 �C and 50 ± 3% RH in a desiccator containing magnesium ni-trate saturated solution [Mg(NO3)2�6H2O] for at least 2 days priorto analysis. Initial gap separation and cross-head speed were setat 50 mm and 1 mm/s, respectively. TS was calculated by dividingthe maximum load at break by initial specimen cross-sectionalarea (ASTM, 2001). EAB was calculated by dividing the extensionat break of the specimen by the initial gage length of the specimen(50 mm) and multiplying by 100 (ASTM, 2001). Each trial was rep-licated at least eight times, and the averages were taken as thedata.

2.7. Water vapor permeability (WVP)

A modification of the ASTM E96-95 gravimetric method (1995)was employed to measure WVP of flexible films (McHugh et al.,1993). Films samples were chosen for WVP testing based on a lackof physical defects such as cracks, bubbles or pinholes. Distilledwater (15 mL) was placed in each test cup to expose the film tohigh relative humidity on one side through a circular opening of4.5 cm diameter. Once the films were secured, the test cups con-taining films were inserted into the pre-equilibrated 0% RH desic-cator cabinets and a fan was placed over the cups to ensuremaintenance of moisture conditions of the films surface. Withintwo hours, steady state had been achieved, the cups were weighedperiodically using an analytical balance (±0.0001 g), five weightswere then taken for each cup at >3h intervals.WVP was calculatedaccording to the WVP method of McHugh et al. (1993).

2.8. Light transmission and opaque

The barrier properties of films against ultraviolet (UV) and vis-ible light were measured according to the method described byLimpisophon et al. (2009), with some modifications. Film opaquewas calculated by the following equation:

Film opaque ðA=mmÞ ¼ �logT=x ð3Þ

where A = absorbance at each wavelength; T = transmittance (%) ateach wavelength; x = film thickness (mm).

2.9. Scanning electron microscopy

The surface of the films was analyzed using a TM-3000 scanningelectron microscope (Hitachi, Japan) in the charge-up reductionmode at an accelerating voltage of 15 kV. The pieces of the filmswere attached to the SEM aluminum sample plate using a dou-ble-sided tape.

2.10. Confocal laser scanning microscopy (CLSM)

CLSM observations of the emulsions were performed on a LeicaTCS SP5 Confocal Laser Scanning head mounted on a Leica DMRE-7(SDK) upright microscope (Leica Microsystems Inc., Heidelberg,Germany) equipped with a 20�HC PL APO/0.70NA oil immersionobjective lens. The various emulsion samples were stained withan appropriate amount of 1.0% (w/v) Nile Blue A (fluorescentdye) in distilled water. The stained emulsions were placed on con-cave confocal microscope slides (Sail; Sailing Medical-Lab Indus-tries Co. Ltd., Suzhou, China), covered with glycerol-coated cover

slips, and examined with a 100 magnification lens and an argon/krypton laser having an excitation line of 514 nm and a HeliumNeon laser (He–Ne) with excitation at 633 nm.

2.11. Modulated differential scanning calorimetry (MDSC)

The glass transition temperature (Tg) and helix–coil transitiontemperature (melting point Tm) of the films were determined byMDSC experiments, using a TA Q100-DSC thermal analyser (TAInstruments, New Castle, DE, USA), according to the method of Belland Touma (1996), with slight modification. The enthalpy (DH) ofthe helix–coil transition was also calculated. Indium was used tocalibrate the equipment. Samples were preconditioned at 25 �Cand 50 ± 3% RH in a desiccator containing magnesium nitrate satu-rated solution for at least 2 days prior to analysis. The films sam-ples (7–8 mg, with 0.1 mg accuracy) were hermetically sealed insolid pans, and an empty pan was taken as the reference. The scanrate was 5 �C/min over an appropriate temperature range: equili-brate at –80 �C, modulate ± 1.0 �C every 60 s, isothermal during5 min, heating at 5 �C/min to 150 �C. The peak temperature of theendotherm on total heat flow profiles was taken as the Tm. The Tg

was characterized on the reversing heat flow signals. The Tg, Tm

and DH were computed from the thermograms by the universalanalyser 2000, version 4.1D (TA Instrument-Waters LLC, New Cas-tle, DE, USA). All experiments were conducted in triplicate.

2.12. Film thickness determination

Film thickness was measured with a digital micrometer (TAIHAIapparatus Co. Ltd., Shanghai, China) to the nearest 0.001 cm. Mea-surements were taken along the length of the specimen five times,and the mean values were used to calculate film tensile strength.

2.13. Statistics

An analysis of variance (ANOVA) of the data was performedusing the SPSS 13.0 statistical analysis system, and a least signifi-cant difference (LSD) with a confidence interval of 95 or 99% wasused to compare the means.

3. Results and discussion

3.1. Characterization of films-forming solution

The size of the lipid particles in the film-forming dispersions(FFD) affects important characteristics of the films, such as theirmechanical and barrier properties. The particle size distributionsof the FFD are plotted in Fig. 1, and the related D3,2 and D4,3 valuesof the FFD are also listed in Table 1. The FFD coded as A in Table 1has no oil in the film formulation. The FFD homogenized by the RShomogenizer showed a bimodal particle size distribution, and twomaxima occurred close to 1 and 7 lm in volume distributions. Thisdistribution changed drastically when the FFD were submitted tomicrofluidization treatment, depending on the microfluidizationpressure or cycles. The FFD homogenized by method ‘‘C’’ or ‘‘E’’exhibited nearly monomodal particle size distributions. Unexpect-edly, the FFD obtained by method ‘‘F’’ recovered a bimodal particlesize distribution (Fig. 1). Those results the moderate microfluidiza-tion treatment (e.g., pressure or cycle) can provide the FFD withnarrower particle size distributions, while high process pressureor cycles made make dispersed lipid particles more susceptibleto flocculation phenomena. Similar results were reported by Jafariet al. (2008), who reviewed the re-coalescence of emulsion drop-lets during high-energy emulsification and pointed out that if the

0.01 0.1 1 10 100 1000

0

2

4

6

8

10

Particle size(µm)

Rel

ativ

e vo

lum

e fr

acti

on (

% v

/v)

B C D E F

Fig. 1. Particle size distribution of film-forming dispersion containing olive oil as afunction of homogenization conditions.

W. Ma et al. / Journal of Food Engineering 113 (2012) 136–142 139

process pressures are too high, they tend to make dispersed parti-cles more susceptible to flocculation phenomena.

The D3,2 and D4,3 of the FFD was a function of the energy input.The D3,2 and D4,3 of the FFM by the RS homogenizer were 3.090 lmand 13.99 lm, respectively. The lipid droplets parameters (D3,2 andD4,3) of the FFD showed a markedly reduced trend when the micro-fluidizer was used in a second step (Table 1). In fact, among theprocess techniques used, the rotor–stator homogenizer providesa lower energy input and so produced the largest particles (method‘‘B’’). Both microfluidization pressure and cycle times affected lipiddroplets distribution in the FFD. When the homogenization pres-sures increase from 69 to 138 Mpa, the D3,2 and D4,3 of the FFDexhibited a distinct trend for different microfluidization cylces. In1 cycle case, the D3,2 and D4,3 of the FFM decreased (methods ‘‘C’’and ‘‘E’’). In 3 cycles case, the D3,2 and D4,3 of FFD, on the contrary,increased significantly (methods ‘‘D’’ and ‘‘F’’). Moreover, the ef-fects of the microfluidization cycles on the lipid droplets distribu-tion were also dependent on microfluidization pressures. Uponincreasing cycles, the D3,2 and D4,3 decreased at the lower pressure(69 MPa), whereas increased at the higher pressure (138 MPa). Themean diameter data further supported that high process pressureor cycles made dispersed lipid particles more susceptible to floccu-lation phenomena due to the incomplete coating of the generateinterfacial surface with the stabilizing polymer (gelatin), giving riseto bridging flocculation.

3.2. Characterization of the films

3.2.1. MC and TSMPackaging films should maintain moisture levels within the

packaged product. Therefore, the knowledge of water solubilityand moisture content of the film is very important for food packag-ing applications. MC and TSM of gelatin–olive oil emulsified filmsas a function of homogenization conditions are shown in Table 2.

Table 2Tensile properties, water vapor permeability and surface hydrophobicity of gelatin films co

Method MC(%) TSM(%) WV

A 19.3 ± 0.8a 44.7 ± 6.1a 5.6B 11.5 ± 0.2d 39.3 ± 2.9b 4.2C 12.2 ± 0.1c 37.5 ± 2.9b 4.6D 12.9 ± 0.4c 33.2 ± 0.6c 4.1E 12.4 ± 0.2c 33.4 ± 0.9c 3.9F 13.64 ± 0.12b 31.8 ± 0.2d 3.7

Values were expressed as means and standard deviations of triplicate measurements.Superscript letters (a-d) indicate significant (P < 0.05) difference within the same column

The MC values significantly decreased due to the inclusion of oliveoil. This is due to the hydrophobic nature of olive oil. Similar re-sults were observed for pistachio globulin protein and fatty acidsemulsified films (Zahedi et al., 2010). Partial protein–water inter-actions replaced with protein–olive oil interactions may accountfor MC reduction occurred in emulsified films. The method Bexhibited the lowest MC in all preparations (Table 2). This phe-nomenon may be attributed to the formation of oil layer on thesurface of emulsified films.

Solubility of a film in water is an important property of ediblefilms, and water insolubility or resistance is usually required fora potentially commercial film. Water solubility is an indication ofa film’s hydrophilicity. After 24 h incubation in de-ionized water,the gelatin–olive oil emulsified films were found to still maintaintheir integrity, while the control (gelatin) film changed its shape.The TSM of emulsified films decreased with increasing pressuresor cycle times (Table 2). Perez-Mateos et al. (2009) also reportedthat the water solution (WS) of cod gelatin films decreased dueto the incorporation of sunflower oil. In contrast, Gontard et al.(1994) found that the addition of lipids to a gluten film causedan increase in its WS. They suggested that the breakage of strongintermolecular bonds in the protein network and the formationof brittle bonds with lipids resulted in structural instability andmade the emulsified film be more susceptible to dissolution. Thepresent results supported that the inclusion of olive oil in gelatinfilms had little effect on their structural integrity incorporation.

3.2.2. Scanning electron microscopy (SEM) and confocal scanning lasermicroscopy (CSLM)

The film surface microstructure, analyzed through SEM, isshown in Fig. 2A. The control (gelatin) films had dense, homoge-neous and smooth structure without grainy and porous structure.The emulsified films had the irregular surface with the distributionof oil droplets, especially the films prepared by the RS homogenizer(Fig. 2A). When the microfluidizer was used in a second step, theirregularity (e.g., caves, holes) of the emulsified films decreasedmarkedly. The application of high pressure homogenization pro-moted relevant changes in the film microstructure even at the low-er pressure (69 MPa, 1 cycle), since differences between bothphases became less marked after the application of the microflui-dization. Lipid particles become embedded in the network andthe smaller particles made them more closely connected withthe polymer. Interestingly, the surface microstructure of the emul-sified film is a function of homogenization conditions. The irregu-larity (e.g., caves, holes) of the films showed a descending trend asthe average lipid droplets diameter (D3,2, D4,3) in the FFD decreasedupon various homogenization treatments (Fig. 1A, Table 1), indi-cating that the lower lipid droplets in the FFD, the less irregularityof the films surface.

The combination of the principles of measuring using fluores-cence and electronic microscopy allows visualizing the morpholog-ical structure of the films, including the distribution of theconstituents in the filmogenic matrix. Fig. 2B shows the CLSM

ntaining olive oil as a function of the homogenization method.

P � 10�10gs�1m�1Pa�1 TS (MPa) EAB (%)

1 ± 0.07a 17.1 ± 2.6b 39.5 ± 7.9c

2 ± 0.0293c 12.0 ± 1.7c 50.5 ± 8.1c

7 ± 0.16b 24.2 ± 2.5a 50.5 ± 4.7c

9 ± 0.04c 21.3 ± 2.8a 106.5 ± 8.3a

6 ± 0.23cd 18.2 ± 1.4b 58.4 ± 9.2b

4 ± 0.11d 23.2 ± 3.0a 106.7 ± 4.7a

.

Fig. 2. Microstructure of gelatin–olive oil emulsified films as a function of homogenization conditions. Panel A: SEM observations of surface of the emulsified films. Panel B:CLSM images of films. Letters a–f within the figures represented the homogenization methods as shown in Table 1.

140 W. Ma et al. / Journal of Food Engineering 113 (2012) 136–142

images of gelatin or emulsifying films. The distribution of olive oil(green) in the film matrix was dependent on the homogenizationtechniques used. CLSM showed a marked decreasing in lipid drop-lets size in films matrix when the microfluidizer was used in a sec-ond step as compared with the films prepared by the RShomogenizer, and the olive oil was non-homogenously distributedas discrete droplets within the continuous gelatin phase (red).

Interestingly, the lipid droplets size of emulsified films pre-sented a similar trend as they did in the FFD upon various micro-fluidization treatments. e.g., when the homogenization pressuresincrease from 69 to 138 Mpa, the lipid droplets decreased for 1 cy-cle case, whereas the lipid droplets increased for 3 cycles case(Fig. 2B). This result was also supported by SEM micrographs(Fig. 1A). CLSM data further indicated that high process pressureor cycles made dispersed particles more susceptible to flocculationphenomena during the drying process of films.

3.2.3. Tensile propertiesTable 2 shows tensile parameters (TS: tensile strength, EAB:

elongation at break) of gelatin films with or without olive oil.The TS and EAB of films was dependent on the special homogeni-zation conditions. The TS of films produced the RS homogenizertreatments decreased as compared gelatin alone films. The reducedcontinuity and cohesion of the protein network due to the large li-pid globules within films matrix might be the reason of this behav-ior. When the microfluidizer was used in a second step, the TS ofthe films increased (Table 2). Since olive oil constituted a non-miscible emulsified phase in the FFD, the protein chains segregatedto form a protein-rich phase. The microfluidization treatment fa-vored protein–protein interactions to a great extent and, as a con-sequence, higher TS were observed. These observations areconsistent with the results of protein–cinnamon oil and gelatin–corn oil films systems (Atarés et al., 2010; Wang et al., 2009).

Table 3Light transmission (%) and transparency (A600/mm) values for the control films and gelatin olive oil films as a function of homogenization conditions.

Sample 200 280 400 500 600 800 A600/mm

A 4.33 ± 0.11a 46.43 ± 1.54a 87.46 ± 0.68a 90.26 ± 0.57a 91.03 ± 0.39a 91.48 ± 0.73a 0.53 ± 0.03f

B 3.10 ± 0.09c 5.80 ± 0.90b 15.11 ± 0.88b 27.68 ± 2.89c 22.38 ± 0.89f 28.22 ± 1.70e 9.85 ± 0.23a

C 4.30 ± 0.11a 2.35 ± 0.28c 8.40 ± 0.42e 19.72 ± 0.54e 28.73 ± 0.31e 40.99 ± 0.96d 6.37 ± 0.06b

D 3.24 ± 0.23c 2.52 ± 0.06c 11.14 ± 0.83d 28.50 ± 0.85c 34.51 ± 1.01c 48.34 ± 1.00c 5.57 ± 0.16d

E 3.23 ± 0.07c 2.56 ± 0.06c 12.52 ± 0.42c 30.90 ± 0.96b 43.30 ± 1.21b 59.82 ± 0.37b 4.54 ± 0.16e

F 3.65 ± 0.12b 3.04 ± 0.13c 15.36 ± 0.42b 25.59 ± 0.65d 32.52 ± 0.89d 41.80 ± 0.72d 5.95 ± 0.15c

LDPEA 0.45 27.65 39.98 45.53 49.81 56.33 4.26

A LDPE, low-density polyethylene (data from Guerrero et al., 2011).

W. Ma et al. / Journal of Food Engineering 113 (2012) 136–142 141

Similar results were also found by Fabra et al. (2009), when addingdifferent lipids to casein films.

The EAB increased as the microfluidization pressure or cycle in-creased, producing more extensible films (Table 2). This effect maybe attributed to the promotion of protein–lipid interactions whenhomogenization conditions were more intense, and some small li-pid particles became embedded in the protein network, whichseemed to have some plasticizing effect on the films. Similar re-sults were observed by Atarés et al. (2010), cinnamon oil had someplasticizing effect on the SPI films, making the films more extensi-ble as the oil content increases.

3.2.4. Water vapor permeabilityThe WVP of the films are shown in Table 2. The WVP signifi-

cantly decreased due to the inclusion of olive oil, regardless ofhomogenization conditions. Unexpectedly, the films prepared bymethod ‘‘B’’ exhibited good water resistance (Table 2). In fact, theRS homogenizer provides a lower energy input and so producedthe largest particles in the related FFD (Table 1) and lipid dropletswith the diameters about 9–30 lm appeared in the CLSM images ofthe films (Fig. 2B). This behavior may be associated with the forma-tion of analogous protein–lipid bi-layer structure, evidenced bySEM data (Fig. 2). In fact, hydrocolloid-lipid bilayer films showedbetter barrier properties (Fabra et al., 2011).

When the microfluidizer was used in a second step, the WVP ofthe films decreased from 5.61 � 10�10 gm�1 s�1 Pa�1 (control) to(3.74–4.67) � 10�10 gm�1 s�1 Pa�1 (emulsified films). Furthermore,the WVP of the films decreased upon increasing microfluidizationpressure or cycle, and the films obtained by method ‘‘F’’ exhibitedthe best water resistance (Table 2). However, high process pressureor cycles made dispersed particles more susceptible to flocculationphenomena, with subsequent formation of the agglomerates of li-pid droplets in the FFD and the obtained films (method ‘‘F’’) (Fig. 1and Fig. 2B). The reason for this abnormal response of WVP to thelipid droplets was unknown. However, this may be associated withthe fact that the intensive microfluidization treatment may en-hance the interactions between oil phase and hydrophobic sectionsof protein molecule due to the high shear stresses developed in themicrochannels of the interaction chamber. This interaction mayweaken the hydrophilic properties of the continuous protein ma-trix, with subsequent decrease in the WVP.

3.2.5. Light absorptionTransparency of films designed for packaging is a relevant prop-

erty since it has a direct impact on the appearance of the coatedproduct. Table 3 shows light transmission values at wavelengthsfrom 200 to 800 nm and transparency for films, in comparison toa commonly used synthetic films (LDPE). Light transmission offilms decreased at almost all wavelengths due to the inclusion ofolive oil, indicating that the films are less transparent. Concomi-tantly, the addition of olive oil produced a sharp increase in thefilm opaque related to the control film (Table 3). However, filmopaque decreased from 9.85 to 4.54–6.37 when the microfluidizer

was used in a second homogenization step (Table 3). Transparencyof films is an auxiliary criterion to judge the compatibility of thecomponents. If compatibility between film constituents is notgood, then the light transmittance is low due to light dispersionor reflection at the two-phase interface (Rhim et al., 2007). Sinceolive oil constituted a non-miscible emulsified phase in the emul-sified films (Fig. 2B), differences between both phases became lessmarked after the application of the microfluidization, withsubsequent decrease in film opaque. Interestingly, film opaquepresented a decreasing trend when the lipid droplets in the FFMand films decreased. Moreover, the opaque values for the filmsprepared by microfluidization techniques were similar or slighthigher than the values of LDPE, a commercial film used for packag-ing purposes (Table 2).

It is also noteworthy that the emulsified films showed excellentbarrier ability to UV light at 280 nm in comparison with the gelatinor LDPE films (Table 2). Some plant protein films (e.g., SPI films)showed excellent barrier properties to UV light in the range of200–280 nm due to the presence of aromatic amino acids (e.g.,tyrosine, phenylalanine and tryptophan) (Li et al., 2004). In con-trast, gelatin films showed poor barrier ability to UV light at280 nm, this may be attributed to the scarcity of aromatic aminoacids in gelatin. Olive oil intake has been associated with variouspositive health benefits in human health due to the presence ofsome antioxidants (López-Miranda et al., 2006) providing withstrong UV absorption. Those results suggested the potential pre-ventive effect of gelatin–olive oil films on the retardation of prod-uct oxidation induced by UV light.

3.2.6. Modulated differential scanning calorimetryFig. 3 shows the typical MDSC thermogram of gelatin films with

or without olive oil. The total heat signal of gelatin film was char-acterized by the presence of an endothermic peak (Tm) at about65.8 �C, attributed to the melting of triple-helix crystalline struc-ture of gelatin. The thermogram for the films with olive oil showedan additional endothermic peak at �8 �C, which can be attributedto the melting peak of olive oil. This is consistent with its non-homogenous incorporation of olive oil into the protein matrix, asevidenced by SEM micrographs and CLSM images (Fig. 2). Concom-itantly, the inclusion of olive oil in films matrix resulted in the in-crease in the Tm of the helix–coil transition of gelatin (Fig. 3A).

Reverse heat flow signals of MDSC were used to characterize theglass transition of the films with or without olive oil. The glasstransition temperature (Tg) of gelatin films was about 61 �C andthis transition temperature tended to increase due to the incorpo-ration of olive oil (Fig. 3B). Bell and Touma (1996) determined theTg of gelatin by separating it from other irreversible thermalchanges using MDSC technique, and reported that its Tg was about60 �C. Since olive oil constituted a non-miscible emulsified phasein the FFD. Thus, the protein chains may segregate to form a pro-tein-rich phase, which may favor protein–protein interactions toa great extent. As a consequence, the emulsified films possessedhigher Tm and Tg in comparison with those of the films without oil.

-50 -25 0 25 50 75 100

-4

-3

-2

-1

0T

otal

hea

t fl

ow (

w/g

)

Temperature (ºC)

G-film RS-film M-film

A

0 25 50 75 100-2.0

-1.6

-1.2

-0.8

-0.4

Rev

ersa

ble

heat

flo

w(w

/g)

Temperature (ºC)

G-film RS- film M-film

B

Fig. 3. DSC profiles of gelatin and gelatin–olive oil films. Panel A: total heat flow.Panel B: reverse heat flow. G-film: gelatin film. RS-film: the film prepared from theFFD by method ‘‘B’’ using a RS homogenizer. D-film: the film prepared from the FFDby method ‘‘D’’ using a rotor–stator homogenizer and a M-110EH microfluidizer.

142 W. Ma et al. / Journal of Food Engineering 113 (2012) 136–142

4. Conclusion

Homogenization techniques including the RS homogenizer andmicrofluidizer were used to prepare filmogenic dispersions. The RShomogenizer provided a lower energy input and so produced thelargest particles, olive oil molecules tended probably to formbi-layer on air side of films. These films exhibited excellent waterbarrier ability, but poor mechanical resistance, extensibility andtransparency. The microfluidizer can provide filmogenic disper-sions with narrower particle size distributions, promotingmechanical resistance, extensibility and transparency of the films.Concomitantly, the WVP decreased upon increasing pressure orcycle. In addition, the obtained gelatin–olive oil films exhibitedexcellent barrier ability to UV light. The transparency values andsurface irregularity of the films presented a reduced trend whenlipid droplets in the FFD and films decreased. Thus, the presentresults indicated that the microfluidizer appeared to be a suitableapproach to modulate lipid droplets distribution of filmogenicmitrix, thus the films’ properites, and to develop edible filmsadapted to a specific target application.

Acknowledgements

The authors thank for the financial support from the NaturalScience Fund of Guangdong Province, China (serial num-ber:10451064101005149), the Fundamental Research Funds forthe Central Universities (SCUT, 2012ZZ0082) and the Open ProjectProgram of State Key Laboratory of Pulp and Paper Engineering,South China University of Technology (No.201023).

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