Stabilized complex film formed by co-adsorption ofb-lactoglobulin and phospholipids at liquid/liquid interface

Yi Zhang, Zhihua An, Guangchen Cui, Junbai Li *

International Joint Lab, Keylab. of Colloid and Intercase Science, The Center for Molecular Science, Institute of Chemistry,

Chinese Academy of Sciences, Zhong Guan Cun, Beijing 100080, PR China

Received 13 May 2002; accepted 28 February 2003

Abstract

A complex film has been formed at a drop surface by the co-adsorption of b-lactoglobulin with the L-a-

dipalmitoylphosphatidylcholine (DPPC), L-a-dipalmitoylphosphatidyl-ethanolamine (DPPE), and L-a-dipalmitoylpho-

sphatidic acid sodium salt (DPPA), respectively, at the water/chloroform interface. By using pendent drop technique we

studied the headgroup effect of lipids on the kinetics of co-adsorption layers. It was found experimentally that a folded

drop surface was formed during co-adsorption and the headgroup of lipids affected on the formation rate of the folded

drop surface. Such a skinlike film demonstrates that there exits an strong interaction between b-lactoglobulin and each

lipid. By depositing one lipid mono- and bilayers onto a solid surface morphology of the mixed lipid/b-lactoglobulin

layers has been investigated by atomic force microscopy (AFM).

# 2003 Elsevier B.V. All rights reserved.

Keywords: Stabilized complex film; b-Lactoglobulin; Liquid/liquid interface

1. Introduction

Mixture of protein and phospholipids remains

widely in nature states such as biological mem-

branes and food emulsion system [1�/6]. In these

systems, the interactions between protein and

lipids occur at the interface. Therefore, the in-

vestigation of the interfacial behavior of proteins

and lipids is of great interest. The study of

adsorption kinetics for such a system is an

important method to learn the information of

system stability and interfacial activity. However,

the possible configuration change of protein as it

adsorbs from bulk to the interface will bring a

difficulty to estimate its effect on the adsorption

activity. b-Lactoglobulin is a whey protein from

milk. Many studies have been done on its adsorp-

tion, reversible and irreversible thermal denatura-

tion as well as the disulfide aggregation properties

[7�/9]. Mackie et al. proved that b-lactoglobulin

could form a monolayer with the 2D network

structure by the co-adsorption with the surfactant,

Tween 20 [10]. In the present work, we have found

out that the b-lactoglobulin itself can form a stable

2D film at a drop surface. Appearance of the

* Corresponding author. Tel.: �/86-10-8261-4087; fax: �/86-

10-8261-2484.

E-mail address: jbli@icas.ac.cn (J.B. Li).

Colloids and Surfaces A: Physicochem. Eng. Aspects 223 (2003) 11�/16

www.elsevier.com/locate/colsurfa

0927-7757/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved.

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skinlike film enforced the drop surface folded. Theco-adsorption of b-lactoglobulin with phospholi-

pids accelerates forming of the folding procedure.

Thus, a stable complex film has been formed in

such a way. The pendent drop technique provides

an ideal method in constructing such a complex by

co-adsorption of the surfactant and protein in two

immiscible solvents, respectively.

2. Experimental section

2.1. Materials

L-a-Dipalmitoylphosphatidylcholine (DPPC),

L-a-dipalmitoylphosphatidyl-ethanolamine

(DPPE), L-a-dipalmitoylphosphatidic acid

(DPPA), b-lactoglobulin were purchased from

Sigma and used without further purification

(99%�/purity). Chloroform with 99% purity was

from ARCOR. By considering the tiny solubility

of chloroform in water the chloroform was satu-rated with Millipore water for all the experiments.

Phosphate was used as a buffer with a concentra-

tion of 10 mM, pH 7.

2.2. Pendent drop technique

The pendent drop setup is the same as described

somewhere else [11]. The pure chloroform or lipid/

chloroform solution was pumped into a pipeline

by a Hamilton pump to the end of a capillary for

producing a pendent drop. The drop was im-mersed into the protein buffer solution. The drop

size can be controlled precisely by a Hamilton

pump via a computer. Few drops were eliminated

and left to the bottom of the cuvette in order to

create a saturated environment and form a fresh

drop.

The capture and analysis of the pendent drop

was based on the commercial software of theaxisymmetric drop shape analysis (ADSA), which

is developed by Neumann and his group [12]. The

forming and detaching time of the drop were

accurately recorded by a video recorder. All

measurements were performed at room tempera-

ture.

2.3. Atomic force microscopy (AFM)

A complex film was prepared by transfering

DPPE mono- or bilayer on a substrate of silicon

wafers with LB technique at a surface pressure of

15 mN m�1 and then to immerse in b-lactoglo-

bulin buffer solution. Rising the LB film with

Millipore water and allowing them to air-dry we

carried out the morphology measurements byAFM. AFM images were achieved using a Nano-

scope IIIa AFM (Digital Instruments, CA) in

tapping mode.

3. Results and discussion

3.1. Adsorption behaviors of pure lipids and b-

lactoglobulin at the chloroform/water interface

From the previous work, we have learned that

pure lipids can form a monolayer by adsorption

into the droplet surface at the water/chloroform

interface [13]. The adsorption obeys a diffusion-

control mechanism. The isotherm of adsorptioncan be better fit by Frumkin model [14], in which

the intermolecular interaction of the adsorption

has been considered at the liquid/liquid interface.

Fig. 1 shows the images of the drop shape

change covered by DPPC adsorption layers with

adsorption time at the concentration of CDPPC�/

1.0�/10�6 M. As one can see that the droplet

volume is reduced at the adsorption time ofaround 2000 s. A neck appears with the elapsed

adsorption time. However, the drop surface is still

smooth.

As b-lactoglobulin solution is used to replace

the pure water, the chloroform and protein aqu-

eous solution produce an immiscible liquid/liquid

interface. Fig. 2 shows the adsorption kinetic

Fig. 1. Images of drops covered by pure DPPC adsorption

layers at the chloroform/water interface at an adsorption time

of: (a) 0; (b) 1200; (c) 2000; (d) 3000 s. CDPPC�/1.0�/10�6 M,

pH 5.

Y. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 223 (2003) 11�/1612

curves of b-lactoglobulin with different concentra-

tion at the chloroform/water interface. As one can

see, at a lower protein concentration, Cb-

lactoglobulin�/0.6 mg l�1, the initial interfacial

tension g0 has the same value of 30.6 mN m�1

as that of pure chloroform/water interface. More-

over, there exists a longer plateau region of around

500 s, which is corresponding to the induction

period of protein adsorption. With the increase of

the protein concentration the induction time is

reduced and finally disappeared at Cb-lactoglobulin�/

2.4 mg l�1. As the protein concentration abruptly

increases to Cb-lactoglobulin�/12 mg l�1, the adsorp-

tion rate of the protein at the liquid/liquid inter-

face becomes extremely fast. The adsorption

reaches to an equilibrium state at around 500 s.

It was experimentally observed that in nearly

every protein concentration the drop volume

decreased with the adsorption time at the chloro-

form/water interface [15]. The corresponding de-

crease of the drop surface area may lead to the

decrease of the surface tension g . However, at a

relatively higher protein concentration, for in-

stance as Cb-lactoglobulin is ]/1.2 mg l�1, the faster

adsorption rate may diminish the influence of the

decrease of the drop area upon the surface tension.

While the drop surface is covered by the

adsorption layer of the pure b-lactoglobulin, at

Cb-lactoglobulin�/1.2 mg l�1, the drop starts to

shrink after 3500 s when the adsorption is closeto a quasi-equilibrium state.

Due to the larger reduction of the drop volume

V , in this experiment over 50% decrease of V , the

interfacial intention value g cannot be calculated

any longer by ADSA. However, an interesting

folded drop surface appeared after 3600 s. The

inner images in Fig. 2 displays the change status of

the drop shape with the adsorption time. Thefolded drop obviously results from the contribu-

tion of protein adsorption layers.

As a control experiment the aqueous b-lactoglo-

bulin solution of Cb-lactoglobulin�/1.2 mg l�1 was

selected for the further study of co-adsorption with

phospholipid. As the lipid DPPC was added in the

chloroform (CDPPC�/1�/10�6 M) the appearance

of the folded drop has been accelerated such thatafter 3000 s a folded drop surface was already

formed. The strong interaction between the lipids

and b-lactoglobulin has been taken into account

from the previous work [16].

3.2. Head group effect of lipids on the formation of

skinlike film

As mentioned above, the addition of DPPC canspeed up the formation of folded skinlike droplet

and increases the strength of the complex film. It

can be deduced that there must be an interaction

between DPPC and b-lactoglobulin molecules at

the interface. Eventually, hydrophobic effect has

been regarded as the main interaction between

lipids and proteins. However, electrostatic inter-

action in some extend may have to be also takeninto consideration if the lipid is charged and

remained at the pH-modified environment. b-

Lactoglobulin has an isoelectronic point at pH

5.1 [17]. It means that the net negative charges in

protein are enriched in the range of pH �/5.1.

Changes in the pH value of the aqueous phase can

only influence the charged state of b-lactoglobulin

molecules. Thus, one may anticipate that pHvariation can slightly change the interaction be-

tween lipid and protein. In fact, while a zwitter-

ionic lipid, DPPE, DPPC or the negatively charged

phospholipid, DPPA is dissolved in the chloro-

form to adsorb at the liquid/liquid interface with

b-lactoglobulin in the immiscible solvent; the co-

Fig. 2. Adsorption kinetic curves of b-lactoglobulin at the

chloroform/water interface with the different concentration of:

(I), Cb-lactoglobulin�0.6; (k), 1.2; ('), 2.4; (%), 4.8; (’), 12 mg

l�1, respectively.

Y. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 223 (2003) 11�/16 13

adsorption rate reflected by the adsorption kineticmeasurements give the predicted results. Fig. 3

shows the adsorption kinetic curves of the three

mixed lipid/b-lactoglobulin systems at the similar

experimental condition. The experimental results

indicate that the adsorption rate of b-lactoglobulin

is in deed becoming faster in the present of lipids

shown by the shifts of the kinetic curves from Fig.

3(a�/d). The mixed DPPA or DPPE/b-lactoglobu-lin system has a relatively faster adsorption

process comparing to that of the DPPC/b-lacto-

globulin system. Both lipids, DPPE and DPPA,

have smaller headgroups and can be charged with

the modification of pH value. Meanwhile, the

headgroup of DPPA or DPPE tends to the

aqueous bulk phase of the protein. And b-lacto-

globulin itself possesses both negative and positivecharges in a molecular chain. Thus, the encounter

of lipid and protein at the interface may neutralize

some charges of proteins. As a consequence, the

slight electrostatic interaction between lipids and

proteins may occur at the interface and lends to a

quicker adsorption rate. However, as one can see

in the Fig. 3 that the kinetic curves of the three-

lipid/protein systems are so close that finally theyreach to the similar equilibrium state. Thus, one

may deduced that the charge interaction between

the lipids and proteins may exist but it is not that

significant.

3.3. Hydrophobic effect on the complex film

The hydrophobic effect is eventually the main

factor to the interaction of lipids and proteins atthe interface. To prove this, we have carried out

the following experiments. A surface hydrophilic-

treated silicon wafer was covered with the lipid

mono- and bilayers by using Langmuir�/Blodgett

dipping technique. We immersed the silicon sub-

strates covered by DPPE mono- and bilayers,

respectively into b-lactoglobulin (Cb-lactoglobulin�/

4.8 mg l�1) solution for 7 h. Then, the sampleswere rinsed with the Millipore water to remove the

rest materials. Two samples of head or tail contact

with b-lactoglobulin have been constructed. Fig. 4

displays the AFM micrographies of protein in the

lipid mono- and bilayers, respectively. In Fig. 4(a),

the flat surface represents the pure lipid bilayer

onto the silicon wafer. After carefully cleaning the

surface the morphology measurements were per-

formed by AFM. With a confocal microscopy by

using a dye NBD-labeled DPPE and AFM we

have proved that the lipid bilayer anchored on the

silica surface. The image of confocal microscopy

shows a smooth green surface by the NBD labeled

DPPE bilayer. However, the resolution of confocal

microscopy is in the order of micrometer andhardly recognizes the organization of the layers.

Thus, the AFM was further used to scan the

surface with the same sample. As shown in Fig.

4(b), after immersing the substrate covered by the

lipid bilayer into b-lactoglobulin solution we have

observed some regular sharp peaks, which repre-

sent the b-lactoglobulin aggregations. The right

hand side is a schematic structure of b-lactoglo-

bulin mixed with DPPE bilayer. The average

height of the peaks is measured as about 3.5 nm,

which is in a good agreement with the thickness of

a protein monolayer [18]. This means that the

protein remains at the surface. Obviously, the non-

isolated protein peaks from the images of the

morphology demonstrate that b-lactoglobulin mo-

lecules well match the DPPE layer and have a

good compatibility. This experiments provide the

evidence for the formation of the above stabilized

lipid/b-lactoglobulin complex film at the chloro-

form/water interface.

Similarly, Fig. 4(c) displays the distribution of

b-lactoglobulin in the pure DPPE monolayer, the

Fig. 3. Kinetic curves of co-adsorption by b-lactoglobulin and

different phospholipid: (a) pure protein without lipids, Cb-

lactoglobulin�/1.2 mg l�1; (b) with DPPC, CDPPC�/1.0�/10�6

M; (c) with DPPE, CDPPE�/1.0�/10�6 M; (d) with DPPA,

CDPPA�/1.0�/10�6 M, respectively.

Y. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 223 (2003) 11�/1614

right hand side gives a schematic structure of b-

lactoglobulin mixed with DPPE monolayer. It is

easily found that there are no sharp peaks of

proteins observed on the top surface. Oppositely,

an around 3.7 nm thick layer with the similar

magnitude of the protein layer was found to cover

onto the DPPE lipid monolayer. The concave

parts represent the defected films, which are

mostly surrounded by the very bright dusts in

the size of 1�/2 mm. Since, the alkyl chains of the

lipids are towards outside of the lipid layer the

hydrophobic chain of the protein is able to directly

Fig. 4. AFM micrographies of the substrate of silicon wafer covered by: (a) pure DPPE bilayer; (b) DPPE bilayer after being immersed

into b-lactoglobulin solution, the right hand side is the schematic structure of DPPE bilayer; (c) DPPE monolayer after being immersed

into b-lactoglobulin solution. Cb-lactoglobulin�4.8 mg l�1 the right hand side is the schematic structure of DPPE monolayer.

Y. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 223 (2003) 11�/16 15

interact with the tails of lipids. This means that thehydrophobic effect between the lipid and protein is

quite significant.

4. Conclusion

A stable lipid/protein complex film can be

constructed by the co-adsorption of lipids and b-lactoglobulin at the chloroform/water interface.

By using pendent drop technique we have deter-

mined the co-adsorption kinetics of lipid and

protein. With different headgroup lipids we have

found that the charged lipids can accelerate the

formation rate of complex film. By depositing

DPPE mono- and bilayers on solid surface and

mixing with b-lactoglobulin we investigated themorphology of the complex film. It is deduced that

the hydrophobic effect between the lipid and

protein is much significant than that of charge

interaction.

Acknowledgements

We acknowledge the financial support of this

research by the National Nature Science Founda-

tion of China (NNSFC29925307), the Major State

Basic Research Development Program (973,

Grant. No. G2000078103), the Chinese Academy

of Sciences and the collaborated project of Ger-man Max Planck Society.

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