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• The two main components of whey Quantification of ␣-lac and -lg in whey proteins/Acacia gum coacervate by capillary gel electrophoresis.
protein isolate, -lactoglobulin and
␣-lactalbumin, undergo complex
coacervation with Acacia gum.
• -lactoglobulin has stronger binding
to Acacia gum than ␣-lactalbumin.
• A residual concentration of proteins
remains in the aqueous phase in all
instances.
a r t i c l e i n f o a b s t r a c t
Article history: Complex coacervation between whey proteins isolate (WPI) and Acacia gum was investigated in order
Received 18 March 2015 to disclose the roles and the contributions of each component of WPI to the formation of the complex
Received in revised form 1 June 2015 coacervate. The main aim was to establish the balance of the main components of WPI, -lactoglobulin
Accepted 2 June 2015
and ␣-lactalbumin, during the phase separation of complex coacervate. The complex coacervation of Aca-
Available online 10 June 2015
cia gum and pure -lactoglobulin, pure ␣-lactalbumin, and WPI have been investigated and compared
together by means of macroscopic observations and capillary gel electrophoresis analyses performed
Keywords:
along pH scans from basic to acidic medium. Coacervate composition was influenced by the pro-
Complex coacervation
Phase separation
tein/polysaccharide (Pr:Ps) ratio and pH. An optimum pH for best coacervation yield was found for each
Capillary electrophoresis Pr:Ps ratio. Non-negligible concentrations of -lactoglobulin and ␣-lactalbumin remain in solution in
Whey proteins isolate most instances. -lactoglobulin undergoes complex coacervation stronger than ␣-lactalbumin in their
Acacia gum competitive coacervation of WPI and Acacia gum.
© 2015 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.colsurfa.2015.06.006
0927-7757/© 2015 Elsevier B.V. All rights reserved.
368 D. Ach et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 481 (2015) 367–374
occurs in case of attractive interaction between a protein and In WPI/AG complex coacervation system three transitions have
a polysaccharide [4–6]. An associative phase separation of elec- been observed in pH scan down to acidic medium. The formation of
trostatic origin between the two polyelectrolytes of opposite soluble complexes was observed first, thereafter phase separation
electrical charges takes place in the case of complex coacerva- took place in a second stage, and the last event was the dissoci-
tion. The complexation of protein and polyelectrolytes is driven ation of the complexes as the isoelectric point of Acacia gum is
by electrostatic interactions; it is controlled by a charge regulation approached [20]. A more detailed picture of complex coacervation
process [7]. has been obtained with the -lg/AG system. It is described as a
The main origin of complex coacervation is an associative nucleation and growth type process where the phase separation is
phase separation phenomenon induced by electrostatic interac- delayed with respect to the formation of the insoluble complexes
tions between at least two macromolecules, typically a protein because a high enough supersaturation is required for nucleation to
and a polysaccharide of opposite charges [8]. But other weak inter- operate [25]. Evidence of six pH-induced structural transitions has
actions, such as hydrogen bonding and hydrophobic interactions, been given during complex coacervation between -lg and Acacia
can contribute to the formation of complex coacervates [9]. The gum at a 2:1 ratio. Characteristic stages are the initiation of the
associative phase separation induces the formation of neutral sol- interactions at pH 4.9, the aggregation of complexes at pH 4.6, the
uble complexes which exhibit an attractive interaction [10]. Such nucleation of phase separation at pH 4.4, the massive phase sepa-
complexes undergo precipitation into liquid droplets that possi- ration at pH 4.2, the observation of large spherical coacervates by
bly settle to form the coacervated phase [11]. Though complex optical microscopy at pH 4.0 and morphological changes of coacer-
coacervation is a phase separation that can be considered as a pre- vates at pH 3.8 [26]. The thermodynamic mechanisms of -lg/AG
cipitation, the complex coacervate particles are extensively swollen binding processes at pH 4.2 include two different binding steps, a
by water and better look like microgel particles in suspension in first exothermic step and a second endothermic step [27].
water. After complexation each biopolymer can be found both in The main objective of the present work was to get a definite bal-
the coacervates and to a small extent, in solution in the aque- ance of the coacervation of the main components of WPI with an
ous phase [12]. Gelatin/Acacia gum polymer pair is one of the anionic polysaccharide, Acacia gum. Commercial WPI was mainly
first and most widely used complex coacervation system [13,14]. composed of -lg and ␣-lac. The precise composition of the com-
Gelatin/Acacia gum coacervates exhibit a wide range of useful func- mercial WPI used for this study was firstly determined. Then the
tionalities for the development of food products such as thickening, formation of complex coacervates was investigated in the WPI/AG
gelling, foaming and emulsifying abilities [15]. Coacervates are also system and compared to that of its pure components in the respec-
used for nano- or microencapsulation of various lipophilic com- tive -lg/AG and ␣-lac/AG systems. The fraction of -lg and ␣-lac
pounds, food ingredients and pharmaceuticals [9,16–18]. involved in WPI/AG complex coacervates as a function of pH and
Whey proteins isolate/Acacia gum (WPI/AG) is another biopoly- biopolymer ratio was determined by capillary electrophoresis.
mer system used for complex coacervation, where WPI is used
to replace gelatin. It was previously shown that the interaction
between WPI and AG is mainly driven by the interaction of - 2. Material and methods
lactoglobulin (-lg) with Acacia gum [19]. Indeed -lg is the major
component of WPI on the one hand; the second major component 2.1. Materials
is ␣-lactalbumin (␣-lac). In addition, the higher isoelectric point of
-lg (pH 5.2) compared to ␣-lac (pH 4.1) leads to the formation of Whey protein isolate (Prolacta® 95) was a gift from Lac-
-lg/AG complexes earlier than the ␣-lac/AG complexes as the pH talis Ingredients (Bourgbarré, France). The powder contained 95%
is lowered from above the isoelectric point [20]. protein with respect to full dry matter. Acacia gum (AG), -
The effect of pH, protein to polysaccharide ratio (Pr:Ps), ionic lactoglobulin (-lg), ␣-lactalbumin (␣-lac), and hydrochloric acid
strength and total biopolymer concentration on complex coacer- were purchased from Sigma–Aldrich. Deionized water of 18 M cm
vates formation has been studied in WPI/AG and -lg/AG systems. was used.
For the WPI:AG system, complex coacervation has been observed
in a pH range from 3 to 5 for mixtures of 10 wt% biopolymers and a 2.2. Preparation of polymers solutions
3:1 Pr:Ps ratio. Only very weak interactions were observed between
the two polymers at higher pH (between pH 5 and 7) [21]. The opti- Whey proteins isolate (WPI), -lg, ␣-lac and Acacia gum were
mum pH of complex coacervation was found at 4.0 for a 2:1 Pr:Ps dispersed in deionized water and mixed under gentle stirring at
ratio. At this pH, phase separation occurred the fastest and the final room temperature until complete dissolution. The concentrations
coacervate volume was the largest. Varying the protein to polysac- of WPI solutions were 10 mg mL−1 for capillary electrophoresis
charide ratio (Pr:Ps) shifted the optimum pH to higher values when assays and 100 mg mL−1 for SDS-PAGE analyses. Complex coacerva-
Pr:Ps was increased. Such shift of optimum pH was probably due to tion studies were carried out with polymer solutions at 10 mg mL−1 .
variations of the concentrations of residual soluble species as the
Pr:Ps ratio was varied. It was also shown that increasing the ionic
strength led to a less concentrated and poorly structured coacervate 2.3. SDS-PAGE
phase, induced by the screening of the electrostatic interactions
[22]. The electrostatic nature of the interactions between -lg and The identification of -lg in WPI was carried out by poly-
Acacia gum has also been pointed out according to the pH depen- acrylamide gel electrophoresis (PAGE) in the presence of sodium
dence of the phase separation, together with the influence of the dodecyl sulfate (SDS). 5 samples of 1–3 L of WPI solution were
protein to polysaccharide ratio and the total biopolymer concentra- deposited in five wells and 10 L of bromophenol blue were added
tion [23]. Finally, changes in -lg/AG complex coacervation were to each samples. Molecular weights were estimated using Fer-
observed in the presence of -lg aggregates. The simultaneous for- mentas electrophoresis protein standard (PageRulerTM Unstained
mation of spherical vesicular coacervates and precipitates were Protein Ladder). The stacking gel and the separation gel were
observed when the -lg solution contained aggregates, whereas 10% acrylamide. After electrophoresis, gels were fixed for 30 min,
only coacervates were observed in aggregate-free -lg solution. destaining was carried out overnight until the background color
Removal of protein aggregates can be performed by centrifugation has completely disappeared, and images of the destained gels were
(RCF = 10,000 × g for 1 h at pH 4.75) [24]. taken.
D. Ach et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 481 (2015) 367–374 369
Fig. 2. WPI solution (5.5%) as a function of pH. Formation of precipitates between pH 5.2 and 4.1.
370 D. Ach et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 481 (2015) 367–374
Fig. 5. WPI/AG complex coacervation as a function of the Pr:Ps ratio (1:1, 2:1 and
4:1) and pH (between 5 and around 2.5). The onset of turbidity is marked by × and
the beginning of phase separation by o.
polymers and polymer precipitation are stronger for polymers of supernatant. Whether there remain non-vanishing concentrations
higher molar mass. But the molar masses of -lg (18.4 kDa) and of anionic and cationic free polymer species seems a matter of a
␣-lac (14.2 kDa) are not so different. In the present case, the elec- large variability. The same competitive coacervation should also
trical charges of the proteins also obviously matter. The charges take place for the anionic partner Acacia gum that contains several
come from anionic and cationic residues of the proteins. According components, arabinogalactan and arabinogalactan-protein com-
to their chemical structures, -lg contains 26 anionic residues (9 plex [36]. It has indeed been disclosed that the negatively charge
aspartic acids, 16 glutamic acids, and one terminal carboxylic acid) polysaccharide arabinogalactan was the main component respon-
and 23 cationic residues (2 tryptophans, 2 histidines, 15 lysines, 3 sible for complex coacervation with -lg [37]. As an outcome, the
arginines, and one terminal amino group) out of 162 residues [33] present results could be used for a better control over the encap-
and ␣-lac contains 16 anionic residues (7 aspartic acids, 8 glutamic sulation process by complex coacervation [38].
acids, and one terminal carboxylic acid) and 21 cationic residues (4
tryptophans, 3 histidines, 12 lysines, 1 arginine, and one terminal Acknowlegments
amino group) out of 123 residues [34]. The densities of ionizable
groups are quite similar for both proteins. At the pie, the proteins Thanks to Prayon S.A. for the financial support to present work.
contain the same linear densities of cationic and anionic residues We are grateful to Mr. Olivier Marcillat (Université Lyon 1, ICBMS
Cpie . Upon shifting the pH from the pie, the linear density of cationic – UMR 5246) for his efficient assistance in SDS-PAGE experimen-
groups increases by x = 10(pie−pH) and the linear density of anionic tations and Mrs. Sandra Balvay (Université Lyon 1, MATEIS – UMR
groups decreases by x = 10(pie−pH) . The total linear charge density 5510) for her help in microplate reading.
was therefore the same for both polymers because the pH shift from
the pie at maximum coacervation were the same and the densities
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