Chai et al., 2010

Original article

Effects of the free and pre-encapsulated calcium ions on the

physical properties of whey protein edible film

Zhi Chai, Jiejing Shang, Yanfeng Jiang, Fazheng Ren & Xiaojing Leng*

CAU&ACC Joint-Laboratory, Key Laboratory of Functional Dairy Science of Beijing and Ministry of Education, College of Food Science &

Nutritional Engineering, China Agricultural University, No.17 Qinghua East Road, Haidian, Beijing 100083, China

(Received 19 February 2010; Accepted in revised form 29 April 2010)

Summary Effects of the free and the pre-encapsulated calcium ions on the physical properties of the whey protein

isolate film were studied for improving calcium content in the edible films. At pH 8, the film-forming process

was hindered by serious protein aggregation and gelation caused by 0.5% (w ⁄w) free calcium ions added in

an 8% whey protein isolate solution. If the calcium ions were pre-encapsulated in the protein microparticles

(contained 17% Ca2+) using spray drying method, and then added in the film-forming solution prepared

using the same protein, the calcium content could be doubled (1%, w ⁄w) without significant effects on the

physical properties of the film. The calcium release ability (55% at pH 1.2, 32% at pH 7.4 and more than

75% with the enzymes) of the film was also investigated.

Keywords Calcium, edible film, encapsulation, mechanical properties, whey protein isolate.

Introduction

Edible films and coatings can extend shelf life andimprove the quality of food by providing barriers tomass transfer. Their matrix is also found to be relevant ascarriers of the functional ingredients as it may be intendedfor the encapsulation and controlled release of anti-oxidants (Han & Krochta, 2007), antimicrobial agents(Zinoviadou et al., 2009) and flavours (Miller &Krochta,1998). Many biomacromolecules including soy, gelatinand whey proteins (WP) have been used for differentpurposes. The latter received a particular attention andare widely used in food products because of their highnutritional value and their ability to form gels, emulsionsor foams (Banerjee & Chen, 1995; Fang et al., 2002;Foegeding et al., 2002). The effects of the driving factorsincluding temperature, pH and concentration on thephysical properties of WP film have been reported inliteratures (McHugh et al., 1993; Anker et al., 1998,1999), and we thus know that the mechanical strength ofthe films reached a maximum with an 8% (w ⁄w) heat-denatured whey proteins isolate (WPI) at pH 8.Nevertheless, a strategic design of ingredient formu-

lation could further improve nutritional functionalitiesof WP, thus increasing applications or creating newapplications in food industry. A clear understanding ofthe structure and formation of protein films is alsorequired to control their properties. Addition of

mineral substance such as calcium, a nutrient havingmany medical and health care uses such as thetreatment of bone loss (Pak & Avioli, 1988; Hughes,1991), can fortify nutritional values of WP films (Fanget al., 2002). As a traditional calcium supplementation,calcium chloride is widely used for its high calciumcontent, good solubility and low price. Its low bio-availability can be improved by binding with WP(Gueguen & Pointillart, 2000; Zhao et al., 2005).However, serious protein aggregation induced by highconcentrated calcium affects the film-forming process(Hongsprabhas & Barbut, 1996; Parthasarathy et al.,1999; Kulmyrzaev et al., 2000; Marangoni et al., 2000).How to avoid such problem and achieve desiredfunctionalities without affecting the physicochemicalproperties of the film is an interesting challenge. Thefocus of this work was to investigate the effects of thefree calcium ions and the WPI pre-encapsulatedcalcium ions on the physical properties of the WPIfilm to find a way to improve the inherit calciumcontent of WPI through the evaluation of the corre-sponding physical properties.

Materials and methods

Materials

WPI (protein > 97%, w ⁄w) is purchased from DaviscoFoods International (Eden Prairie, MN, USA). Glycerol(99% reagent grade), CaCl2, NaOH, NaBr, KBr(analytical grade), ethylenediaminetetraacetic acid

*Correspondent: Fax: +86 10 62737761;

e-mail: [email protected]

International Journal of Food Science and Technology 2010, 45, 1532–15381532

doi:10.1111/j.1365-2621.2010.02303.x

� 2010 Institute of Food Science and Technology

(EDTA), pepsin 3300 units mg)1 (from porcine gastricmucosa, crystalline) and pancreatin 8X (from hogpancreas) are purchased from Sigma Chemical Co. (St.Louis, MO, USA).

Film preparation

A 10% (w ⁄w) WPI stock solution was prepared at roomtemperature and then heat denatured at 80 �C for 30 minin water bath. After cooling to room temperature, asolution containing a certain quantity of CaCl2 orCa2+ ⁄WPIparticles with glycerol as plasticizer was addeddropwise in the WPI solution under gentle agitation. Thefinal WPI concentration was 8% and glyc-erol ⁄WPI = 1 ⁄2 (w ⁄w). The investigated concentrationsof the free Ca2+were from 0% to 1%. The solution wasdegassed in vacuo. The films were cast by applying 5 g ofthe solution evenly onto the polymethacrylate dishes(8 cm diameter) and allowing to dry overnight at roomtemperature. The dry films were equilibrated at22 ± 3 �C and 56% ± 8% relative humidity for at least48 h before subsequently peeled from the casting surfacefor characterisation analyses including mechanical test.Film thickness was determined using a Digimatic Indica-tor (Cheng-Du-Cheng-LiangCo.,China) at ten randomlychosen locations on the films. The average film thicknesswas 90 lm (P > 0.05).

Preparation of the encapsulated calcium

The particle-forming solutions were prepared withCa2+ ⁄WPI mass ratios of 0.2, 0.4, 0.7 and 1 ⁄1 at pH3. The solutions were spray dried using an YC-015Spray Dryer (Ya-Cheng Pilotech Instrument & Equip-ment Co. Ltd, Shanghai, China) with a feed rate of10 mL min)1. The inlet and outlet air temperatures wereset at 180 and 90 �C, respectively. The pre-encapsulatedCa2+ ⁄WPI (PECW) particles were collected at thebottom of the dryer’s cyclone. The quantity ofCa2+encapsulated in the particles was determined usinga traditional EDTA titration method (Bird et al., 1961).The quantity of the PECW particles added in the filmwas described in the main text.

Light scattering measurement

The zeta potential and hydrodynamic size of the parti-cles, d, was determined using a Delsa Nano ParticleAnalyser (Beckman Coulter Inc, Fullerton, CA, USA)based on the principle of dynamic light scattering. Thefluctuations in time of scattered light from particles inBrownian motion are recorded by the autocorrelationfunction G(s) :

G sð Þ ¼ e�sDq2 ð1Þ

D is the diffusion coefficient of the particles, s thedelay time and q the scattering vector; d of the particlesis determined using Stokes-Einstein equation:

D ¼ kT

3pgsdð2Þ

k is the Boltzmann’s constant, T the absolute temper-ature and gs the solvent (water) viscosity.

Colour analysis

Lightness values (L values) of the film-forming solutionvs. [Ca2+] were measured (CIE, 1996) using a ColourDifference Meter TC-P2A (XinAoYiKe Co., Ltd.,Beijing, China) calibrated with a standard white plateprovided by manufacturer.

Mechanical properties

The puncture (PS) and tensile strength (TS) weredetermined according to the method of Peleg (1979)and ASTM D638M (ASTM, 1993) using a textureanalyser (TMS-Pro, Food Technology Corporation,VA, USA) at room temperature.

Water vapour permeability (WVP)

WVP of the films was measured using the modifiedASTM procedure (Gontard et al., 1992):

WVPðgmm=m2d kPaÞ ¼Wx=ATDP

W is the weight gain of the cup (g), x the film thickness(mm), A the area of exposed film (m2), T the time of gain(h) and DP the difference of vapour pressure across thefilm.

Differential scanning calorimeter (DSC)

The fusion temperature of the films was analysed using adifferential scanning calorimeter (DSC Q10, TA, USA)calibrated with pure indium (melting point 156.4 �C).About 10 mg of the film samples was weighed andsealed in an aluminium pan with an encapsulating press.The sample was heated from )125 to 200 �C at a rate of20 �C min)1. An empty sample pan was used as areference.

Controlled release studies

The controlled release study can provide the informa-tion about the film capacity to release the free calciumby the PECW particles in simulated gastric and intes-tinal conditions. Calcium release was determined byincubating an amount of films in an investigated

Effect of encapsulated Ca2+on whey protein film Z. Chai et al. 1533

� 2010 Institute of Food Science and Technology International Journal of Food Science and Technology 2010, 45, 1532–1538

medium with continuous agitation at 37 �C for 6 h(close to the food retention time in human digestiveorgans). The investigated medium included: (i) HClwater solution at pH 1.2; (ii) HCl water solution at pH1.2 with 0.1% (w ⁄v) pepsin; (iii) phosphate-bufferedwater solution (PBS, pH 7.4); (iv) phosphate-bufferedwater solution (PBS, pH 7.4) with 1% (w ⁄v) pancreatin.The quantity of Ca2+released was determined usingEDTA titration (Bird et al., 1961).

Light transmission

The film light transmission was evaluated from 800 to200 nm using a UV Spectrophotometer (Model UV-1100, Rui-Li Analytical instrument Corp., Beijing,China) following the procedure reported by Tang et al.(2005). The transparency was determined as:

T ¼ A600=x

where A600 is the absorbance at 600 nm and x the filmthickness (Perez-Mateos et al., 2009).

Statistical analysis

Analysis of variance (SPSS, version 11.5, SPSS Inc.,Chicago, IL, USA) for a completely random design todetermine the least significant difference at 0.05 was usedto analyse the statistics of the results. Three replicateswere tested at least for each measurement.

Results and discussion

Film-forming solution properties

Figure 1 showed the variation of the WPI aggregate sizevs. the free [Ca2+] at pH 8. The insert image was for thecorresponding autocorrelation functions, G(s). Table 1showed the zeta potential variation of the WPI aggregatevs. [Ca2+] at pH 8. Without addition of the free calcium,the aggregates size was 809 nm (Fig. 1), and the zetapotential was at )23.8 mV. When [Ca2+] increased from0% to 0.5% (domain I), the size decreased from 809 to509 nm (P < 0.05), and the zeta potential varied from)23.8 to )11.6 mV. The decrease in the potentialindicated that the negative surface charges of the protein

were neutralised by the free Ca2+, which led to thedecline of the intermolecular electrostatic repulsion (Ju &Kilara, 1998; Balnois et al., 2000; Marangoni et al.,2000), and made the shrinkage of the aggregates, and themicrostructure became dense. When [Ca2+] continued toincrease from 0.5% to 0.7% (domain II), the potentialdecreased to )6.6 mV, but the size increased to 670 nm.This point suggested that the further charge neutralisa-tion with excess Ca2+led the smaller dense aggregates tojoint together to form larger aggregates. Table 1 showedthe colour analysis as well, where the internal L-valuesincreased rapidly (solution became opaque) when [Ca2+]>0.5%, and we could directly observe the solutionrapidly getting into gel state.

Physical properties

Figure 2 showed the variations of PS and TS of the filmsvs. [Ca2+]. PS and TS without addition of the freeCa2+were 28.1 N mm)1 and 33.7 N mm)2, respectively.When [Ca2+] increased from 0% to 0.1%, PS and TSincreased significantly (P < 0.05) to 54.6 N mm)1 and53.8 N mm)2, respectively; but no more increase

0.0 0.2 0.4 0.6 0.8400

600

800

1000

1200

100 101 102 103 104 105 106 1070.0

0.1

0.2

0.3

0.4

0.5

Domain IIDomain I

d (n

m)

Ca2+ (%)

[Ca2+] = 0

[Ca2+] = 0.1%

[Ca2+] = 0.3%

[Ca2+] = 0.5%

[Ca2+] = 0.7%G(τ

)

τ

Figure 1 Light scattering measurements of whey proteins isolate (WPI)

aggregate size vs. [Ca2+] at pH 8. Insert: autocorrelation functions.

The bars are the standard deviation, and the dash line shows the trend

of variation. The insert image exhibited the aggregation state with

[Ca2+].

Table 1 Variation of the zeta potential and the lightness L-values of whey proteins isolate (WPI) aggregate vs. the free [Ca2+] in the film-forming

solution at pH 8

Ca2+(%) 0 0.1 0.3 0.5 0.7 1.0

Zeta potential (mV)† )(23.8 ± 0.4) )(22.5 ± 0.5) )(17.6 ± 1.1) )(11.6 ± 0.3) )(6.6 ± 1.3) –*

L-value† 0 ± 0.01 0.4 ± 0.02 0.3 ± 0.04 0.6 ± 0.01 2.6 ± 0.09 21.3 ± 1.00

*Gelation not suitable for the test of zeta potential.†Mean ± standard deviation for four replicates.

Effect of encapsulated Ca2+on whey protein film Z. Chai et al.1534

International Journal of Food Science and Technology 2010, 45, 1532–1538 � 2010 Institute of Food Science and Technology

(P > 0.05) for both could be observed when [Ca2+]>0.1%. This observation indicated that the addition of0.1% Ca2+strengthened the crosslink network of theprotein film; however, the excess Ca2+induced complex

protein aggregation and made the film microstructureheterogenous and therefore affected the promotion ofPS and TS (Termonia, 1990).Figure 3 showed the variations of the water vapour

permeability (WVP) vs. [Ca2+], where WVP was kept atabout 20 g mm m)2 d kPa and no significant variationscould be observed with the free [Ca2+] (P > 0.05).Similar data were also reported by Fang et al. (2002).The concentrated Ca2+reinforced the crosslink networkof the film, reduced the intervals between the proteinmolecules, and was expected to decrease WVP; however,the hygroscopic salt also enhanced the hydrophilicityand hygroscopicity of the system and favoured the watermolecules to permeate into the matrix. Therefore, thefinal WVP values were the results of the balance of theabove opposite effects.

PECW particle properties

Figure 1 indicated that the quantity of the free calcium inthe film-forming process could not exceed a certaincritical value. The key to improve the inherit calciumcontent of WPI is how to control the activity of thecalcium ions in the system, i.e. if the activity of the calciumions is pre-limited into the tiny particles rather than theentire solution, the large-scale aggregation in the solutioncould be avoided. Table 2 showed the state of the film-forming solutions vs. the quantity of the added PECWparticles. ThePECWparticles used in thiswork contained17%Ca2+(Ifmore than 17%, thePECWparticles becamemoist and difficult to collect from the dryer’s cyclone).The maximum quantity of the PECW particles added inthe film-forming solutionwas 0.5% (w ⁄w), which allowedhaving 1% (w ⁄w)Ca2+in the film.Obviously, if the dryingconditions in the particle preparation process could beimproved, the calcium content in the film could be furtherenhanced. Considering the point of using the encapsula-tion method in this work was to increase the calciumcontent in the film, only the film containing the maximumquantity of the calcium through PECWmethod was thusused to do the comparison.

DSC analysis

Figure 4 showed the thermograms of three differentWPI films: without Ca2+, with 0.4% free Ca2+or withPECW particles. The fusion temperature (Tf) of thefilm without Ca2+was at 130 �C (denoted using the

0.0 0.2 0.4 0.6 0.8 1.00

10

20

30

40

Film containing Ca2+-WPI particles

Ca2+ (%)

WV

P (

g m

m 2

4h–1

m–2

kpa

–1)

Figure 3 Variation of the water vapour permeability (WVP) of the

whey proteins isolate (WPI) films vs. [Ca2+]. The PECW particle–

incorporated WPI film is denoted with the arrow. Mean ± standard

deviation for three replicates.

20

40

60

80

100

0.0 0.2 0.4 1.0

20

30

40

50

60

70

TS

(N

mm

–2)

PS

(N

mm

–1)

Film containing Ca2+-WPI particles

Ca2+ (%)

Figure 2 Mechanical properties (PS and TS) of the whey proteins

isolate (WPI) films vs. [Ca2+]. Solid symbol: PS; open symbol: TS. The

pre-encapsulated Ca2+ ⁄WPI (PECW) particle–incorporated WPI film

is denoted with the arrow. Mean ± standard deviation for three

replicates.

Table 2 State of the film-forming solution and the total Ca2+ content in the film vs. the quantity of the pre-encapsulated Ca2+ ⁄WPI (PECW)

particles added in the film-forming solution

PECW particle quantity (%, w ⁄ w) 0 0.3 0.5 0.7 1.0

Film-forming solution state Solution Solution Solution Gel Gel

Ca2+ (%, w ⁄ w) 0 0.6 1.0 1.3 2.0



maximum value of the peak). The broad shape of thefusion peak was mainly because of the incorporatedwater in protein matrix. When the free Ca2+or thePECW particles were added, Tf of the films increased to142 and 138 �C, respectively. Tf was related to the heatstability of the protein microstructure. The stronger thecross linkage of the network structure was, the higher Tf

the film had. The increase in Tf of the sample with freeCa2+indicated that the cross linkage of the networkstructure was enhanced. In contrast, the increase in theTf of the PECW particle–incorporated WPI film wasweaker. Figure 5 schematically illustrated the effectsof the free Ca2+and PECW particles on the microstruc-ture of the WPI films. High concentration of freeCa2+could induce serious protein aggregation andmade the matrix dense. If Ca2+was pre-encapsulated,the cations mobility was restricted in particle matrix(more details were shown in Figure 6), and the extent ofaggregation in the whole system could be largely reduced.

Calcium-controlled release studies

Figure 6 compared the Ca2+-release capacity of thePECW particles alone (a) and the PECW particle–

incorporated WPI film (b). For the PECW particles(Fig. 6a), more than 65% Ca2+could be released fromthe particle matrix at pH 1.2 but less than 50% at pH 7.4in 6 h. The higher release capacity at acid environmentwas attributed to the electrostatic repulsions betweenCa2+and the positive charges of the protein. For the film(Fig. 6b), the Ca2+release was reduced (55% at pH 1.2

Figure 5 Schematic image of the Ca2+ effect on the microstructure of whey proteins isolate (WPI) films.

0 1 2 3 4 5 6

0

20

40

60

80

100

Time (h)

pH = 1.2pH = 1.2+pepsinpH = 7.4pH = 7.4+pancreatin

Rel

ease

of

Ca

( )

Rel

ease

of

Ca

( )

(a)

(b)

0 1 2 3 4 5 6

0

20

40

60

80

100

Time (h)

pH = 1.2pH = 1.2+pepsinpH = 7.4pH = 7.4+pancreatin

Figure 6 Comparison of the Ca2+-release capacity of the PECW

particles alone (a) and the PECW particle–incorporated whey proteins

isolate (WPI) film (b).

Figure 4 Differential scanning calorimeter (DSC) thermograms of the

three different whey proteins isolate (WPI) films.



and 32% at pH 7.4 in 6 h), because the proteinmolecules in the film matrix hindered the movement ofthe Ca2+released from the particle matrix. In the use ofthe digestive enzymes, the Ca2+could be largely released(more than 75% with pepsin and more than 95% withpancreatin in 6 h), and no significant difference betweenthe particle system and the particle-incorporated filmsystem was observed. The higher release capacity withthe pancreatin was caused by its stronger ability tohydrolyse the protein molecules.

Light transmission and transparency

Figure 7 compared the light transmittance and trans-parency (insert image) of the films without the freeCa2+(a) and with the PECW particles (b), and nosignificant difference was found between both systems(P > 0.05). The two light transmittance curves werealmost superimposed and both showed about 80%transmittance between 400–800 nm while less than 6%transmittance between 200–300 nm. The UV absorptionwas chiefly because of the aromatic side chains astryptophan, phenylalanine, and tyrosine in protein(Goldfarb et al., 1951). PS, TS and WVP of the PECWparticle–incorporated WPI film were inserted in Figs 2and 3, respectively. In these figures, the differencesbetween the films without Ca2+and with the PECWparticles were not significant (P > 0.05).

Conclusions

Taking into account the divalent cations as calciuminducing serious protein aggregation and thus prevent-

ing the formation of film, the quantity of calciumadded to WPI films could not exceed a criticalconcentration. Using a pre-encapsulation method, thecritical concentration of calcium could be exceededwithout significant variation of the physical propertiesincluding PS, TS, WVP and transparency of the filmunder the present experimental conditions. This meth-od could potentially be used to incorporate the otherminerals or organic substances that could cause proteinaggregations.

Acknowledgments

This work was supported by China High-Tech (863)project (2007AA10Z311), and National Science andTechnology Support Programme (2006BAD04A06).

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0 200 400 600 800 1000

0

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A B0.0

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Tra

nspa

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Chai et al., 2010