Improved water vapor barrier of whey protein films by addition of an acetylated monoglyceride

Improved water vapor barrier of whey protein films by addition of an acetylated monoglyceride

Innovative Food Science & Emerging Technologies 3 Ž2002. 81᎐92 Improved water vapor barrier of whey protein films by addition of an acetylated monogl...

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Innovative Food Science & Emerging Technologies 3 Ž2002. 81᎐92

Improved water vapor barrier of whey protein films by addition of an acetylated monoglyceride Martin Anker, Jonas Berntsen, Anne-Marie Hermansson, Mats StadingU SIK ᎏ The Swedish Institute for Food and Biotechnology, P.O. Box 5401, SE-402 29 Goteborg, Sweden ¨ Received 2 April 2001; accepted 17 September 2001

Abstract This study aimed to determine to what extent the water-vapor barrier of whey protein isolate ŽWPI. films could be improved by adding a lipid and make laminate and emulsion films. The laminate whey protein᎐lipid film decreased the water vapor permeability ŽWVP. 70 times compared with the WPI film. The WVP of the emulsion films was half the value of the WPI film and was not affected by changes in lipid concentration, whereas an increased homogenization led to a slight reduction in WVP. The mechanical properties showed that the lipid functioned as an apparent plasticizer by enhancing the fracture properties of the emulsion films. This effect increased with homogenization. The maximum strain at break was 117% compared with 50% for the less-homogenized emulsion films and 20% for the pure WPI films. Phase-separated emulsion films were produced with a concentration gradient of fat through the films, but pure bilayer films were not formed. 䊚 2002 Elsevier Science Ltd. All rights reserved. Keywords: Whey protein; Acetylated monoglyceride; Laminate films; Emulsion films; Microstructure; Water vapor permeability; Mechanical properties; Edible films Industrial rele¨ ance: Biopolymeric films usually have good mechanical properties but their barrier property - due to their hydrophilic character ᎐ against water vapor is poor. The aim of this study was to produce composite whey protein ᎐ lipid films Žlaminate and emulsion films. to improve the barrier properties against water vapor. The data showed that laminate whey protein ᎐ lipid films decreased the water vapor permeability 70 times. A future challenge will be to control phase separation of emulsion films and to create completely phase-separated bilayer films.

1. Introduction The barrier properties of biopolymeric films are important parameters when considering a suitable barrier in foods and food packaging. Protein and polysaccharide films are generally good barriers against oxygen at low and intermediate relative humidity ŽRH. and have good mechanical properties, but their barrier against water vapor ŽWV. is poor. This poor barrier is due to

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Corresponding author. Tel.: q46-31-335-56-00; fax: q46-31-8337-82. E-mail address: [email protected] ŽM. Stading..

their hydrophilic character. In many applications, a better barrier against WV is preferable since low levels of water activity must be maintained in low-moisture foods to prevent texture degradation and to minimize deteriorative chemical and enzymatic reactions ŽKester & Fennema, 1986.. One way to achieve a better WV barrier is to add an extra hydrophobic component, e.g. a lipid, and produce a composite film. Here, the lipid component serves as the barrier against WV, whereas the protein or polysaccharide film provides the barrier against oxygen and gives the necessary strength. A composite film made of a protein or polysaccharide and a lipid can be divided into laminates Žin which the lipid is a distinct layer within or atop the biopoly-

1466-8564r02r$ - see front matter 䊚 2002 Elsevier Science Ltd. All rights reserved. PII: S 1 4 6 6 - 8 5 6 4 Ž 0 1 . 0 0 0 5 1 - 0

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M. Anker et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 3 (2002) 81᎐92

meric films. and emulsions Žin which the lipid is uniformly dispersed throughout the biopolymeric film.. Both the laminate and emulsion films offer advantages. The laminate films are easier to apply with regard to the temperature, due to the distinct natures of the support matrix and lipid ŽKoelsch, 1994.. During the casting of the lipid onto the protein or polysaccharide film, the temperatures of the film and lipid can easily be controlled separately. When producing the emulsion films, the temperature of the emulsion must be above the lipid-melt temperature but below the temperature for gelation and solvent volatilization of the structural network. The main disadvantage of the laminated films, however, is that the preparation technique requires four stages: two casting and two drying stages. This is why the laminated films are less popular in the food industry despite their good barrier against WV ŽDebeaufort & Voilley, 1995.. The preparation of the emulsion films requires only one casting and one drying stage, but the finished films are still rather poor barriers against WV, since the water molecules still permeate through the non-lipid phase. Therefore, it would be interesting to form a bilayer film from the emulsion mixture where the lipid is concentrated at the surface during the drying process. Hence, a good barrier against WV would be achieved with a less time-consuming preparation method. This phase separation is, however, difficult to accomplish. Many researchers have examined the WVP and mechanical properties of composite films made from proteins or polysaccharides with added lipids. For example, composite protein᎐lipid films had lower WVP values than control protein films from caseinates ŽAvenaBustillos & Krochta, 1993., whey protein ŽBanerjee & Chen, 1995; Berntsen, 2000; McHugh & Krochta, 1994a,b; Perez-Gago & Krochta, 1999., zein ŽWeller, ´ Gennadios & Saraiva, 1998., and wheat gluten ŽGennadios, Weller & Testin, 1993; Gontard, Duchez, Cuq & Guilbert, 1994.. Accordingly, the barrier against WV was improved with composite polysaccharide᎐lipid films made from methylcellulose ŽDebeaufort & Voilley, 1995; Greener & Fennema, 1989a,b; Kester & Fennema, 1989a,b; Koelsch & Labuza, 1992. and hydroxypropyl methylcellulose ŽHagenmaier & Shaw, 1990; Kamper & Fennema, 1984a,b.. The reduced migration of moisture in bicomponent foods has also been studied. Kamper and Fennema Ž1985. used a bilayer film consisting of lipid and a layer of hydroxypropyl methylcellulose between tomato paste and ground crackers. The film substantially slowed transfer of water from the salted tomato paste to the crackers. Composite films have also been used to retard dehydration of coated foods. Avena-Bustillos, Cisneros-Zevallos, Krochta and Saltveit Ž1994. showed that using an edible coating of caseinate ᎐lipids on peeled carrots reduced surface dehydration and white blush formation. This is important

since white blush on the surface is a major factor reducing consumer acceptance of minimally processed carrots. Another example is frozen king salmon that was coated with an edible whey protein᎐lipid solution. The reported moisture loss was reduced by 42᎐65% during three weeks of storage at y23 ⬚C ŽStuchell & Krochta, 1995.. There are many lipids that have been used as WV barriers in composite films. Examples include different waxes, fatty acids, and acetylated monoglycerides ŽAMG.. In the present study, an AMG with a melting point of 35 ⬚C was used since it would be able to melt in the mouth. Furthermore, the lipid should preferably be commercially available in large amounts and be reasonably priced for industrial applications. The aim of the present study was to produce composite whey protein᎐lipid films Žlaminate and emulsion films. to improve the barrier against WV. The focus of the emulsion films was to study the effect of the lipid concentration and homogenization. Moreover, phase separation between the protein and the lipid was investigated to obtain bilayer films.

2. Materials and methods 2.1. Materials Whey protein isolate ŽWPI. was obtained from Arla Foods ŽVidebaek, Denmark.. WPI ŽLacprodan DI-9224. is a functional WPI used for protein fortification of clinical nutrition products and as sports foods. The WPI powder had a dry content of 93 " 2% protein Ž N = 6.38., 0.2% fat, 0.2% lactose, max 6.0% moisture, and max 4.0% ash, with a pH between 6.5 and 7.0 Ž0.10% solution.. The concentration of proteins in the WPI powder was 74% ␤-lactoglobulin, 18% ␣lactalbumin, 6% bovine serum albumin, and 2% immunoglobulins Žvalues supplied by manufacturer .. For further details regarding the molecular mass and cysteine groups of each protein, see Anker, Stading and Hermansson Ž1999.. Glycerol ŽG. was used as plasticizer and was obtained from BDH Laboratory Supplies ŽPoole, England.. Grindsted Acetem 70-00 ŽAcetem. was used as an additive to the WPI films to improve the barrier against WV. The lipid was obtained from Danisco Ingredients ŽBrabrand, Denmark. and is an acetic acid ester of monoglycerides made from edible, fully hydrogenated lard in which 70% of the free hydroxyl groups have been acetylated. 2.2. Experimental layout The experimental layout is summarized in Table 1. The WPI and Acetem films were produced as reference films to compare to the results from the laminate and

Table 1 Experimental layout Conc. of WPIa w% Žwrw.x

Amount of Acetemb Žg.

Ratio Acetemr WPIc

Temp WPId

Temp Aceteme Ž⬚ C.

Temp finalf Ž⬚ C.

level 1 Žrev.rmin.

time 1 Žmin.

level 2 Žrev.rmin.

Time 2 Žmin.

level 3 Žrev.rmin.

time 3 Žmin.

WPI Acetem Laminate

10.8 ᎐ 10.8

᎐ 3.8 3.8

᎐ ᎐ 0.4

᎐ ᎐ 70

᎐ 145 195

76.5 ᎐ 76.5

᎐ ᎐ ᎐

᎐ ᎐ ᎐

᎐ ᎐ ᎐

᎐ ᎐ ᎐

᎐ ᎐ ᎐

᎐ ᎐ ᎐

Emulsion films Emul A Emul B Emul C Emul D

ᎏ effects of lipid concentration 10.8 43.2 10.8 21.6 10.8 10.8 10.8 5.4

4 2 1 0.5

70 70 70 70

80 80 80 80

76.5 76.5 76.5 76.5

500 500 500 500

2.5 2.5 2.5 2.5

13 000 13 000 13 000 13 000

1 1 1 1

᎐ ᎐ ᎐ ᎐

᎐ ᎐ ᎐ ᎐

Emulsion films Emul E Emul F Emul G

ᎏ effects of homogenization 10.8 5.4 10.8 5.4 10.8 5.4

0.5 0.5 0.5

70 70 50

80 80 60

76.5 76.5 76.5

500 500 ᎐

2.5 2.5 ᎐

16 000 19 000 19 000

1 0.5 0.5

22 000 24 000 24 000

2 1.5 4.5

Emulsion films Emul H Emul I Emul Jg

ᎏ effects of phase separation 10.8 43.2 10.8 43.2 10.8 21.6

4 4 2

70 70 72

80 80 110

76.5 76.5 76.5

500 500 500

2.5 2.5 0.5

᎐ ᎐ ᎐

᎐ ᎐ ᎐

᎐ ᎐ ᎐

᎐ ᎐ ᎐

a

The ratio of WPIrG s 2.1 was constant in all film formulations. Amount of added Acetem. c Amount of Acetem related to the initial concentration Žamount. of WPI. d Temperature of the WPI solution when Acetem was added. e Temperature of Acetem when added to the WPI solutions. f Final temperature of the heated film-forming solutions. g The cast film-forming solution was placed in an oven for 40 min at 50 ⬚C. b

Homogenization

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Film

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M. Anker et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 3 (2002) 81᎐92

emulsion films. Only one technique for producing the laminate film was tested, since the main interest in this study was focused on the emulsion films. The emulsion films were investigated in three series. In the first series ŽEmul A᎐D., the lipid concentration was varied to study if an increased lipid concentration reduced the WVP. In the second series ŽEmul D᎐G., higher degrees of homogenization were applied in order to obtain a high tortuosity, since increased tortuosity is hypothesized to reduce WVP by increasing the diffusion path for the water molecules. In the third series ŽEmul H᎐J., the homogenization was minimized to induce a phase separation between the added lipid and the protein matrix. The purpose was to obtain a coalesced lipid layer dried in contact with air and a concentration gradient of lipid through the film to the opposite surface, which was to consist of pure protein, and thereby obtain a bilayer film. All films produced with the different techniques were preconditioned in a climate room at 23 ⬚C and 50% RH for at least 48 h before testing. 2.3. Film formation 2.3.1. WPI films The concentration of the WPI films was 10.8% Žwrw. WPI based on the dry weight. G was added as a plasticizer, and the ratio of WPIrG s 2.1 was held constant. The WPI aqueous solutions were mixed for 1 h, adjusted to pH 7.0 with 1 M sodium hydroxide, degassed for 10 min, and heated in oil bath to 76.5 ⬚C at a heating rate of f 6 ⬚Crmin. A 15.4-g solution was applied to each polystyrene Petri dish Ž⭋ 14 cm. to minimize thickness variations. To ensure that the films produced could be peeled intact from the casting surface, the Petri dish was covered with a hydrophobic surface of Teflon FEP film ŽNorton Performance Plastics Corp., Akron, OH, USA.. The cast solutions were allowed to cool and dry at room temperature for f 4 h and were then dried in a climate room at 23 ⬚C and 50% RH for 16 h. For further details see Anker, Stading and Hermansson Ž1998.. 2.3.2. Acetem films Acetem Ž3.8 g. was heated to 145 ⬚C and cast on a heated Ž45 ⬚C. aluminum plate with a frame of Teflon Ž⭋ 11 cm.. After 5 min, the film was peeled off. 2.3.3. Laminate films The WPI films were made according to the method described previously. Acetem Ž3.8 g. was heated to 195 ⬚C and cast on the WPI film. The high temperature was chosen to reduce the viscosity of the lipid and thereby facilitate spreading on the WPI film. The laminate film was cooled for 10 min and peeled off.

2.3.4. Emulsion films The WPI film-forming solution described above was used as a base solution in all emulsion film-formulations. The cast emulsion solutions were allowed to cool and dry at room temperature for f 4 h and were then dried in a climate room at 23 ⬚C and 50% RH for 16 h if not stated otherwise. 2.3.5. Effect of lipid concentration (Emul A᎐D) In the first series the concentration of lipid was varied. The lipid was heated to 80 ⬚C and added to the WPI solution in the water bath at a temperature of 70 ⬚C. At the same moment, the velocity of a magnetic stirrer was set to 500 rev.rmin. When the temperature had reached 76.5 ⬚C after 2.5 min, the solution was taken out of the oil bath and was further homogenized for 1 min at 13 000 rev.rmin with an Ultra Turrax T25 basic mixer ŽIKA Labortechnik, Janke & Kunkel, Staufen, Germany.. The solution was degassed for 1 min and then cast. 2.3.6. Effect of homogenization (Emul D᎐G) In the second series, the homogenization was increased in order to obtain smaller and more numerous particles. The lipid was heated, added to the WPI solution, and homogenized Ž2.5 min, 500 rev.rmin. in the same way as for the first series. The solution was then taken out of the oil bath and was further homogenized for 1 min at 16 000 rev.rmin and for 2 min at 22 000 rev.rmin ŽEmul E.. The homogenization of Emul F was 0.5 min at 19 000 rev.rmin and 1.5 min at 24 000 rev.rmin. Thereafter, both solutions were degassed for 1 min before film casting. In Emul G, a different heat treatment was applied, which made it possible to increase the homogenization time. The WPI solution was taken out of the oil bath when the temperature had reached 50 ⬚C, which is below the denaturation temperature 66.5 ⬚C of ␤-lactoglobulin at pH 7 ŽHegg, 1980., and the homogenization started when the lipid heated to 60 ⬚C was added. The solution was homogenized for 0.5 min at 19 000 rev.rmin and subsequently for 4.5 min at 24 000 rev.rmin. Homogenization at 24 000 rev.rmin increased the temperature by ; 6 ⬚Crmin which limited the homogenization time, since the final temperature was kept constant at 76.5 ⬚C. The films were degassed for 1 min and subsequently cast. 2.3.7. Effect of phase separation (Emul H᎐J) In the third series, the homogenization was reduced to induce a phase separation between the added lipid and the protein matrix. Emuls H and I were made using the same method described for the first series ŽEmul A᎐D., but without the homogenization for 1 min at 13 000 rev.rmin and without degassing. Degassing cools the solution quickly and thereby makes it

M. Anker et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 3 (2002) 81᎐92

more difficult to accomplish a phase separation. For Emul J, the level of homogenization was minimized, and the effect of retarded cooling was investigated. The lipid was heated to 110 ⬚C and added to the 72 ⬚C WPI solution. After 0.5 min at 500 rev.rmin, the final temperature of 76.5 ⬚C was reached. The films were cast and placed in an oven at 50 ⬚C for 40 min, and due to the higher drying temperature, 20 g of solution was used instead of 15.4 g. A higher amount of solution was used to get a film of sufficient thickness that could separate in the two different phases. Accordingly, the drying time in the climate room increased to 40 h.

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an Ultracut E ultramicrotome ŽReichert Jung, Wiena, Austria.. Sections were picked up on copper grids and stained with 5% uranyl acetate Ž1 h. and 0.3% lead citrate Ž1 min.. The microstructure of the film crosssections was observed at 100 kV Ž=3597. using a LEO 906E transmission electron microscope ŽLEO Electron Microscopy Ltd., Cambridge, England. with a high-performance, low-intensity charged-coupled device ŽCCD. camera. The preparation of each experimental point was reproduced once, since our previous paper ŽAnker, Stading & Hermansson, 2000. showed the technique to be reliable and that one reproduction was enough for accurate micrographs.

2.4. Transmission electron microscopy 2.5. Film thickness Small filmstrips, f 5-mm long and 3-mm wide, were cut from the films, and small triangular incisions were made at the end of each filmstrip to be able to distinguish between the top Ždried in contact with air. and the bottom Ždried in contact with Teflon. of the films. Filmstrips were fixed for 2 h in 37% formaldehyde fumes and thereafter post-fixed for 2 h in 2% osmium tetraoxide fumes. The samples were then dehydrated for 15 min each in a graded ethanol series: 50%, 75%, and three times at 99.5%. Treated filmstrips were then embedded in increasing concentrations of acrylic resin ŽLR White Resin Medium Grade; Standard Supplies AB, Kallered, Sweden.: 2 h in 50% concentration, ˚ overnight in 75%; f 8 h in 100%, overnight in 100%; and f 4 h in 100%. Subsequently, the polymerization of the resin proceeded at 60 ⬚C for 20 h. Ultrathin sections, ; 90 nm, were cut on a diamond knife using

Before testing, the thickness of the films was measured by a digital micrometer Žmodel IDC-112CB; Mitutoya Corp., Tokyo, Japan. at the center and at four positions around the perimeter for the WVP measurement and at five positions along the rectangular strips for the mechanical properties. The WVP and the mechanical properties were calculated using the average thickness for each film replicate and measured in the climate chamber prior to all measurements. 2.6. Water ¨ apor permeability WVP was determined according to ASTM E96-90 Ž1990. and corrected for the stagnant air gap inside test cups for hydrophilic films using the WVP Correction Method ŽMcHugh, Avena-Bustillos & Krochta, 1993..

Table 2 Summary of results Film

WVPa Žgrmmrm2 h kPa.

MCa Ž%.

Thicknessa Ž␮m.

Ea ŽMPa.

␴b a ŽMPa.

WPI Acetem Laminate

13.8" 1.7 0.2" 0.1 0.2" 0.1

18.6" 0.4 0.8" 0.1 9.7" 0.8

140 " 5 298 " 14 368 " 17

96 " 3.7 1 " 0.2 36 " 3.3

2.2" 0.11 0.02" 0.01 1.0" 0.08

20 " 3 13 " 4 29 " 5

Emulsion films ᎏ effects of lipid concentration Emul A 7.7" 0.8 Emul B 6.5" 0.6 Emul C 7.1" 0.6 Emul D 7.4" 0.2

11.7" 0.3 15.0" 0.5 16.9" 0.3 18.6" 0.5

432 " 26 292 " 17 224 " 10 207 " 6

7 " 0.3 23 " 0.9 34 " 5.0 52 " 4.6

0.3" 0.01 0.8" 0.03 1.0" 0.08 1.2" 0.15

29 " 2 23 " 4 42 " 4 50 " 2

Emulsion films ᎏ effects of homogenization Emul E 5.8" 0.5 Emul F 6.4" 0.3 Emul G 7.1" 0.6

21.0" 0.9 22.2 " 0.7 20.9" 0.4

205 " 14 207 " 6 186 " 7

50 " 7.4 56 " 1.7 40 " 1.1

1.6" 0.11 1.9" 0.06 1.3" 0.03

117 " 17 99 " 10 23 " 3

Emulsion films ᎏ effects of phase separation Emul H 8.8" 0.9 Emul I 7.2" 1.4 Emul J 8.2" 1.4

11.0" 0.3 15.0" 0.6 15.3" 1.0

439 " 15 266 " 19 323 " 17

8 " 0.2 27 " 1.2 28 " 2.7

0.3" 0.01 0.9" 0.03 0.8" 0.06

19 " 3 27 " 4 32 " 6

a

Mean values " 95% confidence interval.

␧b a Ž%.

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M. Anker et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 3 (2002) 81᎐92

A climate room set at 23 ⬚C and 50% RH was used to test WVP. Distilled deionized water was placed in the bottom of the test cups to expose the film to a high RH inside the cups. The films were mounted with the surface dried in contact with air Žlipid-rich. facing the high RH. At least eight replicates of each experimental point were evaluated. Due to the stagnant air gap inside the cups, the RH on the underside of the films varied between 67 and 100%. Consequently, the gradient varied between 50 and 67% and 50 and 100%. For further details regarding the WVP measurements, see Anker et al. Ž1998.. 2.7. Moisture content (MC) MC was determined by drying small filmstrips in an oven Žmodel T6060; Heraeus, Molndal, Sweden. at ¨ 105 ⬚C for 24 h. Small test specimens were cut and put on glass Petri dishes, and the weights before and after the oven drying were recorded. MC was calculated as the percentage of weight loss based on the original weight, in accordance with ASTM D644-94 Ž1994.. At least 13 replicates of each experimental point were evaluated Žeight for the Acetem film.. 2.8. Mechanical properties A texture analyzer Žmodel TA-XT2; Stable Micro Systems, Godalming, England. was used to determine mechanical properties in accordance with ASTM D88291 Ž1991.. The films were tested in a climate room at 23 ⬚C and 50% RH. The initial grip separation and crosshead speed were set to 50 mm and 24 mmrmin, respectively. The tested filmstrips were 80-mm long and 6-mm wide. Force and elongation were recorded during extension, and Young’s modulus Ž E ., stress at break Ž ␴ b ., and strain at break Ž ␧ b . were calculated. E and ␴ b were calculated using the initial cross sectional area. At least 20 replicates of each experimental point were evaluated.

3. Results and discussion The results from the WVP, MC, thickness, and mechanical properties for the different film formulations are summarized in Table 2. 3.1. The WPI, Acetem and laminate films 3.1.1. Microstructure The micrograph from the reference WPI film is shown in Fig. 1. The protein network in the laminate film does not differ in structure compared with the WPI film and is, thus, not presented. One might have expected a different protein structure at the surface on

Fig. 1. Representative transmission electron micrograph of the center of the WPI film. Dark areas represent the protein matrix and the white areas the water and plasticizer phase.

the laminate film compared with the WPI film, due to the cast lipid layer. An idea was that the surface of the WPI film would melt in contact with the hot lipid during casting and that the lipid would penetrate through the surface of the WPI film. However, no traces of such penetration of lipid could be seen in the micrographs, and, consequently, the lipid layer does not seem to influence the microstructure of the WPI film. 3.1.2. WVP The WVP of the WPI, Acetem, and laminate films is shown in Table 2. The WVP value of the WPI film is as high as 13.8 g mmrm2 h kPa, which is due to the hydrophilic character of whey proteins. The Acetem film and the laminate film both have a WVP of 0.2 g mmrm2 h kPa, which is 70 times lower than the value for the WPI film. The fact that the laminate film has the same WVP value as the Acetem film indicates that the resistance of the WPI film to WV is negligible compared with Acetem. Similar results were observed for a methylcellulose film coated with anhydrous milk fat where the permeance of the methylcellulose film was 200 times higher than the laminate film ŽShellhammer & Krochta, 1997b.. This is in agreement with an electrical analogy, the resistance to moisture transfer of the lipid-coated film is the sum of the resistances of the individual layers ŽBarrer, 1968.. Improved WV barrier by laminate films has been reported in numerous studies for zein ŽLai & Padua, 1998., wheat gluten ŽGontard, Marchesseau, Cuq & Guilbert, 1995., methylcellulose ŽGreener & Fennema, 1989a,b; Park, Testin, Park, Vergano & Weller, 1994. and hydroxypropyl methylcellulose ŽKamper & Fennema, 1984a,b.. Zein films coated with a sorghum waxrmedium-chain triglyceride showed a WVP value of 0.1 g mmrm2 h kPa compared with 9 g mmrm2 h kPa for the uncoated zein film ŽWeller et al., 1998..

M. Anker et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 3 (2002) 81᎐92

This improvement Ž; 100 times. is comparable with the improvement Ž70 times. shown in the present study. 3.1.3. MC The WPI film contains 18.6% water whereas the Acetem film shows an MC of 0.8% ŽTable 2.. Because of the almost equal contribution of weight of the two layers, the laminate film has an MC value of 9.7%, which falls between the two reference films. 3.1.4. Mechanical properties In Table 2 it is seen that the Acetem film has a negligible mechanical strength compared with the WPI film. The stress at break Ž ␴ b . for the Acetem film is ; 1% of the value of the WPI film, whereas the laminate film shows behavior affected by both the WPI and Acetem film. The ␴ b value is 1.0 MPa compared with the 2.2-MPa value for the WPI film. The reduced value is due to the lack of structural integrity of the lipid layer. The force divided by the film area Žthickness = width. calculates the stress, and since the lipid layer contributes only to an increased thickness, the stress decreases. The result from the strain at break Ž ␧ b . does not show the same tendency. It is seen that the laminate film can be stretched 29% before breaking, the Acetem film 13%, and the WPI film 20% ŽTable 2.. These results indicate that the lipid layer could function as a plasticizer for biopolymeric films, and similar results have been reported for zein films ŽLai & Padua, 1998., egg albumen films ŽGennadios, Weller, Hanna & Froning, 1996., and methylcellulose films ŽPark et al., 1994.. For example, a methylcellulose film laminated with a palmitic acid-corn layer increased the elongation from 28 to 65% ŽPark et al., 1994.. 3.2. Emulsion films ᎏ effects of lipid concentration (Emul A᎐D) 3.2.1. Microstructure The micrographs of the Emul A᎐D films ŽFig. 2. show a clear reduction in lipid globule size when the

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lipid concentration is reduced. The Emul A film in Fig. 2a shows large globules of fat and the Emul B film shows similar characteristics. In the Emul C film, the lipid globules are smaller and some rodlike particles are seen, which are much smaller than the other particles. The Emul D film in Fig. 2d shows a protein network that is less dense Žincreased grayness. compared with the other films in the series. This is due to the decreased lipid concentration. The fat-to-protein ratio decreases from 4 to 0.5 Žby a factor of 8., whereas the thickness decreases only from 432 to 207 ␮m Žby a factor of 2. for the Emul A and D films. Thus, the relative protein volume increases in the Emul D film, and a less dense, more porous structure can be formed. Furthermore, the lipid globules’ variation in size increases in the series Emul A᎐D. The larger variation in size in the Emul D film compared with the Emul A film can be explained by the lesser tendency of lipid globules to coalesce when the lipid concentration is low. 3.2.2. WVP There is no significant difference in the WVP for the Emul A᎐D films ŽTable 2.. Shellhammer and Krochta Ž1997a. found a non-linear increase in WVP when the lipid concentration was reduced in whey protein films. When beeswax and anhydrous milk fat concentration decreased to 35 and 40% of the whey protein, respectively, a sharp increase in WVP occurred. These concentrations correspond to that in the Emul D film, and an increase in WVP could have been expected. However, no such change was observed, which might be due to the lipid used in this study having different properties and the experimental conditions not being the same. Further reduction of lipid concentration is needed to find the minimum concentration of lipid. This result is interesting from an economical point of view, especially when considering an industrial application. The lipid can be added with a low fat-to-protein ratio Žlipidrproteins 0.5. and still give a similar barrier as for a higher fat-to-protein ratio Žlipidrproteins 4.. A low fat-to-protein ratio, and thus a low lipid

Fig. 2. Representative transmission electron micrographs of the center of Ža. Emul A; Žb. Emul B; Žc. Emul C; and Žd. Emul D films Ž=3597.. Dark areas represent the protein matrix and the white areas the lipid phase.

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concentration, also results in better mechanical properties compared to a high fat-to-protein ratio. In the present study, the WVP of the emulsion films was half the value of the WPI film. Similar results have been reported for various emulsion films, such as caseinates ŽAvena-Bustillos & Krochta, 1993. and whey protein ŽBanerjee & Chen, 1995; McHugh & Krochta, 1994a,b; Perez-Gago & Krochta, 1999.. For example, ´ an acetylated monoglyceridercaseinate s 0.25 film had a WVP of 1 g mmrm2 h kPa compared with 1.5 g mmrm2 h kPa for the pure caseinate film ŽAvenaBustillos & Krochta, 1993.. This illustrates that emulsion films are still rather poor barriers against WV, since the water molecules still permeate through the non-lipid phase. 3.2.3. MC There is an almost linear increase in MC when the lipid concentration is reduced ŽTable 2.. The Emul A film ŽAcetemrWPI s 4. has an MC of 11.7%, which increases to 18.6% for the Emul D film ŽAcetemrWPI s 0.5.. This increase is due to the constant MC of the protein and the lipid. When the lipid concentration is decreased, the effect of the low MC of the Acetem diminishes, and the MC approaches that of the protein. 3.2.4. Mechanical properties In Table 2, it is clearly seen that ␴ b and ␧ b decrease when the lipid concentration increases. The decreased ␴ b value when the lipid concentration increases is in agreement with the findings of other researchers ŽWeller et al., 1998; Shellhammer & Krochta, 1997a.. Weller et al. Ž1998. stated that the decrease in ␴ b accompanying the increase in lipid concentration was related to the weakening effect of lipid on the protein network, due to the lack of structural integrity of the lipid. The ␧ b is higher for the Emul D film than for the WPI film ŽTable 2.. This indicates that the incorporated lipid has a plasticizing effect.

3.3. Emulsion films ᎏ effect of homogenization (Emul D᎐G)

3.3.1. Microstructure The average fat droplet size is larger in the Emul D film compared with the Emul E film, which has the same fat content ŽFig. 3a,b.. The latter also has a higher number of globules. These effects are due to the increased homogenization of the Emul E film. Furthermore, the protein structure seems to be denser in the Emul E film. The differences between Emul E and Emul F are only minor, since the film preparation technique differs only slightly in homogenization. The size of lipid particles in the Emul G film look similar to those in the Emul E and Emul F films, but the border between the lipid and the protein is less pronounced. One could speculate that this could be indicative of an increased interaction between the lipid and the protein, which was obtained by adding the lipid and start the homogenization below the denaturation temperature of ␤-lactoglobulin. In the other emulsion films, the lipid was added after the denaturation of the protein, and it is believed that the protein in those films already had started to form a protein network, resulting in reduced interaction ability between protein and lipid. Fig. 4 shows that different structures are formed at the top and bottom, compared with the center. This is illustrated with the Emul D and E films. ŽThe Emul F and G films show the same characteristics as the latter.. The lipid droplets are more spherical in the center of the films compared with the flatted form at the top and bottom. This is due to the different drying process. At the top, the evaporation and diffusion of water is extensive, whereas the lower mass transfer of water in the center results in a slower drying rate, and consequently, a slower gelation of the protein matrix. Thus, the fat globules are compressed at the top, whereas they can attain a more spherical form in the center. At the bottom, the drying rate may be less important, but

Fig. 3. Representative transmission electron micrographs of the center of Ža. Emul D; Žb. Emul E; Žc. Emul F; and Žd. Emul G films Ž=3597.. Dark areas represent the protein matrix and the white areas the lipid phase.

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the flattened lipid globules are most probably a result of the pressure of the total volume of the film solution. Furthermore, a more porous protein structure was formed in the center of the Emul D film, compared with the denser structure at the top and bottom ŽFig. 4a.. Similar results were seen for the WPI in the present study and WPI films from our previous investigation ŽAnker et al., 2000.. This is due to the slower drying rate, allowing the protein matrix to form a more aggregated structure, which leads to the formation of pores. This effect cannot be seen for the Emul E film, and consequently, a more compact structure is formed throughout the film. 3.3.2. WVP The Emul E film shows a slightly lower WVP value compared with the Emul D film ŽTable 2.. This lower WVP value is due to the increased diffusion path for the water molecules when the barrier contains more numerous and smaller lipid globules. This result is in agreement with the findings of McHugh & Krochta Ž1994b., who found that decreasing emulsion-particle

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diameters correlated well with linear decreases in WVP. Further homogenization in the Emul F and G films does not lead to further decreases in WVP. 3.3.3. MC There is a slight increase in the MC for the more homogenized Emul E᎐G films compared with the Emul D film ŽTable 2., but the difference is relatively small due to the constant lipidrprotein ratio. 3.3.4. Mechanical properties Table 2 shows the ␴ b and ␧ b values for the Emul D᎐G films. The ␴ b increases with homogenization in the series Emul D᎐F. The maximum ␧ b was 117% for the Emul E film compared with 50% for the Emul D film ŽTable 2.. The increased ␧ b is most probably related to the greater ability of the smaller and more numerous fat globules to dissipate fracture energy and prevent crack propagation, and thereby delay the break. Thus, the lipid functions as an apparent plasticizer by enhancing the fracture properties of the emulsion films, and this effect increases with homogenization. The

Fig. 4. Representative transmission electron micrographs of the Emul D and E films at the top, center, and bottom Ž=3597.. Dark areas represent the protein matrix and the white areas the lipid phase. The schematic film thickness is representative of the examined films.

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reduced ␴ b and ␧ b for the Emul G film, however, are related to the different film-formation techniques. The homogenization of the lipid started below the denaturation temperature of ␤-lactoglobulin. This probably resulted in fewer protein᎐protein interactions and more protein᎐lipid interactions, and since the former affects the aggregation of the protein matrix and the subsequent gelation of the film-formation solutions, the mechanical properties are reduced. 3.4. Emulsion films ᎏ effect of phase separation (Emul H᎐J) 3.4.1. Microstructure The micrographs from the top, center, and bottom of the Emul H film ŽFig. 5b. illustrate the effect of the phase separation and are compared with the corresponding micrographs from the Emul A film ŽFig. 5a. Žwith the same lipid concentration .. The micrographs show large differences in microstructure, and the only variance in film preparation between the films is the degree of homogenization. In the Emul A film, several globules can be distinguished on the micrographs, and

there is a variation in globule size. This characteristic is seen through the entire film Žtop, center and bottom.. In the Emul H film, a concentration gradient of fat globules was formed due to flocculation and partial coalescence. The top image of the Emul H film shows two lipid globules that have partly coalesced, but with protein still present between the droplets. The micrograph of the center shows a similar situation, and the bottom image shows a layer consisting of a pure protein phase. When this image is compared with the image of the bottom for the Emul A film, it is obvious that a phase separation has occurred. However, a pure bilayer structure is not obtained. The Emul I and J films show a phase separation similar to the Emul H film, and hence, is not shown. The only difference between the Emul H and Emul I᎐J films is the location of the phase frontier. In the Emul H film, the frontier between the lipid and protein phases lies nearer to the bottom than in the Emul I᎐J films. This difference is related to the higher lipid concentration in the Emul H film. No effect of the minimized homogenization and the retarded film

Fig. 5. Representative transmission electron micrographs of the Emul A and H films at the top, center, and bottom Ž=3597.. Dark areas represent the protein matrix and the white areas the lipid phase. The schematic film thickness is representative of the examined films.

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cooling on the phase separation in the Emul J film was observed. 3.4.2. WVP No significant differences are seen between the Emul A and H films ŽTable 2.. Nor is any improvement observed in the WVP of the Emul I᎐J films compared with the Emul B film ŽTable 2.. Thus, the phase separation in the Emul H᎐J films does not improve the barrier against WV compared with the other emulsion films, since the water molecules still permeate through the non-lipid phase. 3.4.3. MC The MC is very well correlated with the lipid concentration discussed previously: when the lipidrprotein ratio is constant the MC is unchanged. Consequently, the MC of the Emul A and H films Žlipidrproteins 4. is constant at ; 11%, and the MC of Emul I᎐J films and the Emul B film Žlipidrproteins 2. is unchanged Ž; 15%.. 3.4.4. Mechanical properties In Table 2, the ␴ b and ␧ b values of the Emul A and H films are presented. The ␴ b values are the same and the only difference is the slightly increased ␧ b of the Emul A film. The Emul I᎐J films show the same ␴ b and ␧ b values as the Emul B film. The increased ␴ b for the Emul B and I᎐J films compared with the Emul A and H films is due to the lower relative concentration of lipid.

4. Conclusions This study showed that laminate whey protein᎐lipid films decreased the WVP 70 times. The WVP of emulsion whey protein᎐lipid films was half the value of the pure whey protein films and was not affected by changes in lipid concentration, whereas an increased homogenization led to a slight reduction of the WVP. The tensile tests showed that the lipid functioned as an apparent plasticizer by enhancing the fracture properties of the emulsion films, and this effect increased with homogenization. Phase-separated emulsion films were produced in this study with a concentration gradient of fat through the film, but pure bilayer films were not formed. Further research is needed to fully understand the factors that control the phase separation so that a completely phase-separated bilayer film can be produced.

Acknowledgements We thank Annika Altskar ¨ for her skilful technical

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assistance with cutting and staining of the embedded film samples for the transmission electron microscope and for valuable discussion regarding the results. We thank Stable Micro System ŽGodalming, England. for supplying the TA-XT2 texture analyzer. The Swedish Board for Technological and Industrial Development ŽNUTEK. supported this work, as did Arla Foods, Orkla Foods, StoraEnso Research, and Tetra Pak. References Anker, M., Stading, M., & Hermansson, A.-M. Ž1998.. Mechanical properties, water vapor permeability, and moisture contents of ␤-lactoglobulin and whey protein films using multivariate analysis. Journal of Agricultural and Food Chemistry, 46, 1820᎐1829. Anker, M., Stading, M., & Hermansson, A.-M. Ž1999.. Effects of pH and the gel state of the mechanical properties, moisture contents, and glass transition temperatures of whey protein films. Journal of Agricultural and Food Chemistry, 47, 1878᎐1886. Anker, M., Stading, M., & Hermansson, A.-M. Ž2000.. Relationship between the microstructure and the mechanical and barrier properties of whey protein films. Journal of Agricultural and Food Chemistry, 48, 3806᎐3816. ASTM Ž1990.. Standard test methods for water vapor transmission of materials. Designation: E96-90. In ASTM, Annual Book of ASTM Standards Žpp. 834᎐841.. Philadelphia, PA. ASTM Ž1991.. Standard test methods for tensile properties of thin plastic sheeting. Designation: D882-91. In ASTM, Annual Book of ASTM Standards Žpp. 182᎐190.. Philadelphia, PA. ASTM Ž1994.. Standard test method for moisture content of paper and paperboard by oven drying. Designation: D644-94. In ASTM, Annual Book of ASTM Standards Žpp. 1᎐2.. Philadelphia, PA. Avena-Bustillos, R. J., & Krochta, J. M. Ž1993.. Water vapor permeability of caseinate-based edible films as affected by pH, calcium cross-linking and lipid content. Journla of Food Science, 58, 904᎐907. Avena-Bustillos, R. J., Cisneros-Zevallos, L. A., Krochta, J. M., & Saltveit, M. E. Ž1994.. Application of casein᎐lipid edible film emulsions to reduce white blush on minimally processed carrots. Posthar¨ est Biological Technology, 4, 319᎐329. Banerjee, R., & Chen, H. Ž1995.. Functional properties of edible films using whey protein concentrate. Journal of Dairy Science, 78, 1673᎐1683. Barrer, R. M. Ž1968.. Diffusion and permeation in heterogeneous media. In J. Crank, & G. S. Park, Diffusion in Polymer Žpp. 165᎐217.. New York: Academic Press. Berntsen, J. Ž2000.. Improved water vapor barrier of whey protein films. Diploma thesis, Chalmers University of Technology, Sweden. Debeaufort, F., & Voilley, A. Ž1995.. Effect of surfactants and drying rate on barrier properties of emulsified films. International Journal of Food Science and Technology, 30, 183᎐190. Gennadios, A., Weller, C. L., & Testin, R. F. Ž1993.. Modification of physical and barrier properties of edible wheat gluten-based films. Cereal Chemistry, 70, 426᎐429. Gennadios, A., Weller, C. L., Hanna, M. A., & Froning, G. W. Ž1996.. Mechanical and barrier properties of egg albumen films. Journal of Food Science, 61, 585᎐589. Gontard, N., Duchez, C., Cuq, J.-L., & Guilbert, S. Ž1994.. Edible composite films of wheat gluten and lipids: water vapor permeability and other physical properties. International Journal of Food Science and Technology, 29, 39᎐50. Gontard, N., Marchesseau, S., Cuq, J.-L., & Guilbert, S. Ž1995.. Water vapor permeability of edible films of wheat gluten and

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