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Complex Coacervation of Milk Proteins with Sodium Alginate Elham Ghorbani Gorji, Abdul Waheed, Roland Ludwig, Jose Luis Toca-Herrera, Gerhard Schleining, and Sara Ghorbani Gorji J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03915 • Publication Date (Web): 28 Feb 2018 Downloaded from http://pubs.acs.org on March 1, 2018

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Journal of Agricultural and Food Chemistry

ζ= 9.73

3.0 pHopt

ζ= 6.97

Absorbance at 400nm

2.5

ζ= -39.66

2.0 pHφ2 1.5

pHφ1

ζ= -50.76

1.0 0.5

pHC

0.0 0

1

2

3

4

5

6

7

pH

Sodium alginate ( ) 0.1% wt – bovine serum albumin ( ) 0.2% wt

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Complex Coacervation of Milk Proteins with Sodium Alginate

1 2 3

Authors: Elham Ghorbani Gorjia, Abdul Waheedb, Roland Ludwig a, José Luis Toca-Herrerac,

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Gerhard Schleininga and Sara Ghorbani Gorjid

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Affiliations:

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a

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(BOKU), Vienna, Austria

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b

9

c

Department of Food Science and Technology, University of Natural Resources and Life Sciences

Faculty of Agriculture, University of Hohenheim, Stuttgart, Germany

Institute for Biophysics, Department of Nanobiotechnology, University of Natural Resources and

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Life Sciences Vienna (BOKU), Muthgasse 11, A-1190 Vienna, Austria

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d

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Australia

The University of Queensland, School of Agriculture and Food Science, Brisbane 4072, QLD,

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Abstract

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Beta-lactoglobulin (BLG) and bovine serum albumin (BSA) coacervate formation with sodium

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alginate (ALG) was investigated by turbidimetric analysis, zeta potential, particle size, viscosity,

16

transmission electron

17

measurements as a function of pH (1.0-7.0) and, protein:alginate mixing ratio (1:1, 1.5:1 and 2:1,

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1:0 and 0:1 w/w). Critical pH values of phase transitions for BSA-ALG complexes (pHC, pHɸ1,

19

and pHɸ2) representing the formation of soluble and insoluble complexes of a protein-ALG

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mixture (2:1) at pH 4.8, pH 4.2 and pH 1.8, respectively. In case of BLG-ALG, Critical pH values

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(pHC, pHɸ1, and pHɸ2) were found to be 4.8, 4.2 and 1.6, respectively. The pHopt values, expressed

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by the highest optical density, were pH 2.8 for BSA-ALG and 2.4 for BLG-ALG. TEM and zeta

23

potential results showed that maximum coacervate formation occurred at pH 4.2 for both protein-

24

polysaccharide solutions. The interaction between BLG-ALG and BSA-ALG was spontaneous

25

exothermic at pH 4.2 according to ITC measurements. The findings of this study provide insights

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to have a thorough understanding about the nature of interactions between milk proteins and ALG

27

and formulate the new applications for food, pharmaceutical, nutraceutical and cosmetics

28

applications.

microscopy (TEM) and isothermal titration calorimetric (ITC)



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1. INTRODUCTION

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Proteins and polysaccharides as natural functional ingredients are extensively used in the food,

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biomedical industries and pharmaceutical 1. The manipulation of the macromolecular interactions

32

between these biopolymers is a crucial element in their application for different purposes

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Protein-polysaccharide complexation occurs from an electrostatic interaction between the

34

oppositely charged biopolymers in an aqueous phase 5. This attractive interaction can take place in

35

either a single phase (soluble) or in two phase (insoluble) 6. Complex coacervation is the

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formation of insoluble complexes which leads to a liquid-liquid macroscopic phase separation,

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with solvent rich and complex rich phases

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order to decrease the free energy of the system 8. The electrostatically driven formation of

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complexes, can be influenced by several physicochemical parameters in the system i.e. type of

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biopolymer, biopolymer concentration, mixing ratio, biopolymers reactive groups, size, pH,

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charge density, temperature, and ionic buffer strength 9,10.

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The interaction of proteins and polysaccharide gained considerable attention during the last decade

43

according to their usability in different areas such as food, cosmetic industries and pharmaceutical,

44

where they were used as bioactive delivery devices, fat replacers, gels, emulsion stabilizer, edible

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films, coatings and for food texturisation 11,12. The effects of biopolymer ratio, total concentration

46

and pH changes were assessed for the β-lactoglobulin-acacia gum system. Being investigated by

47

Weinbreck et al., the effect of biopolymers charge density on their interaction on complex

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formation between whey protein isolate and two other types of polysaccharides, λ- carrageenan

49

and a lactic bacteria exopolysaccharide lead to a precipitate in the complexation of whey protein

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isolate- λ- carrageenan because of the high charge density of the polysaccharide 9,13.

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Alginate is an anionic, linear and water-soluble polysaccharide extracted from three different

52

species of brown algae and is consist of varying amounts of 1–4 linked α-L-guluronic and β-D-

5,7

2–4

.

. The aggregation of soluble complexes occurs in


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mannuronic acid residues with pKa values of 3.4 – 4.4. The sequence of monomers and the

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molecular weight of this biopolymer specifies its physical properties 14,15. Alginate is mainly used

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in the food, pharmaceutical, and cosmetic industries due to its special characterisation such as

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biocompatibility, biodegradability, gelling properties, immunogenicity, non-toxicity, and

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relatively low cost 16. Studies showed that alginate is able to go through complex coacervation or

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precipitation with several proteins like pea protein, albumin, whey protein, β-lactoglobulin, canola

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protein, and gelatin A. The formed coacervates and precipitates depend on the type of protein but

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also pH, total biopolymer concentration, mixing ratio, and ionic strength17,18. Bovine serum

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albumin (BSA) is a highly soluble globular protein that is found in whey, milk, and plasma and

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has 585 amino acid residues, and a molecular weight of 66.5 kDa19. It contains a high number of

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amino acids with acidic side chains (203 aspartates and glutamates) and with basic side chains

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(164 lysines, arginines and histidines), most of them surface exposed which results in an

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isoelectric point of 5.0. The secondary structure of this protein is mainly composed of α-helices

66

(66%) with a remaining content of β-sheets turn and side chains (34%) 20. Convenient structural,

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physicochemical, and biological properties makes BSA a proper model protein for pharmaceutical,

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cosmetic and food systems

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acid residues

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aspartates and glutamates) and with basic side chains (18 lysines, arginines and histidines), most

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of them surface exposed which results in an isoelectric point of 5.2. This protein is highly

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functional with thermal aggregation, gelation and surface-active properties 23. These two proteins

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are frequently used as model proteins in the development of pharmaceutical, nutraceutical,

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cosmetic, and food formulations 24. They have electro negative hydrophilic domains at pH values

75

above their isoelectric points and an excess of positive charges below pH of isoelectric points and

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this characteristic make them to be capable of forming complexes with various polyelectrolytes,

22

21

. β-lactoglobulin is a small, globular whey protein with 162 amino

. It contains also a high number of amino acids with acidic side chains (27



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especially with potent charge polysaccharides 25. Electrostatic interactions lead the association and

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the phase behaviour in the protein/polysaccharide complexes. The formation, the structure, and the

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property of the complex are affected by physicochemical conditions like pH, ionic concentration,

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intrinsic features, distribution of charges, size, stiffness of polysaccharide chains, etc.9,13.

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The objectives of current study are to characterize the interactions occurring between the proteins

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BSA and BLG and the anionic polysaccharide ALG in aqueous solutions. Moreover, the effect of

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solvent pH and biopolymer mixing ratio on complex formation involving milk proteins with ALG

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was determined. The interactions between these biopolymers in solution have been studied using

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analytical techniques, including spectroscopy, size measurements, zeta potential, isothermal

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titration calorimetry and different microscopy techniques. Each of these techniques will provide

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some information on the nature of the interaction between these biopolymers in a broad range of

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pH values.

89 90

2.

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2.1 Sample preparation

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β-Lactoglobulin and bovine serum albumin were obtained from Sigma (Germany). Guluronate-

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rich sodium alginate (Protanal® LF 10/60 Sodium Alginate) was kindly provided by FMC

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Biopolymers. 0.2% wt BSA and BLG solutions were made separately in Milli Q water (ultrapure

95

water). The solutions were stirred for 30 min and kept in 4°C for 12 hours. 0.1% wt ALG solution

96

was prepared by mixing sodium alginate in Milli Q water and stirring overnight (12h). The

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mixtures of BSA-ALG and BLG-ALG were prepared by dissolving each protein solutions with

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ALG solution.

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measurements and 2:1 for other measurements were prepared.

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2.2 Determination of critical pH values by spectrophotometry

MATERIAL AND METHODS

Mixing ratios of Protein:ALG 0:1, 1:1, 1.5:1, 2:1 for spectrophotometry


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Protein and sodium alginate dispersions at different protein-ALG ratios of 0:1, 1:1, 1.5:1, 2:1 and

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1:0 were prepared using the stock solutions. The prepared dispersions were acidified gradually

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(from pH 7 to 6.80, 6.60, 6.40 to 1) by addition of 0.05 M, 0.1 M, 0.2 M and 2 M HCl solution

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under magnetic stirring at each pH value. The turbidity (optical density) of proteins and sodium

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alginate dispersions mixtures as a function of pH from ~7 to ~1 was measured using a UV/visible

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light spectrophotometer (UV 1800 Shimadzu, Japan) at maximum absorbance wave length

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obtained before for the solutions (~400 nm). Measurements were done in cuvettes (plastic, 1 cm

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path length) and for the blank reference Milli-Q water was used. ALG, BSA and BLG solutions

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were used as controls at 0.1% wt concentration. The critical pH values i.e. pHC: development of

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soluble complexes, pHφ1: development of insoluble complexes, pHopt: maximum optical density,

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pHφ2: dissolution of complexes (Table 1) were calculated based on the intersection point of two

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curve tangents.

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2.3 Zeta potential and size measurements

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The electrophoretic mobility of samples was detected using a Malvern Zetasizer NS 200 apparatus

115

(Malvern Instruments, Germany) fitted with a DTS1070 sample cell. This instrument applies a

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photon correlation spectroscopy (PCS) technique to measure particle size in constant random

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thermal, or Brownian motion. Before the measurement, the sample solutions were diluted to 100

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to 500 times with ultra-pure water to avoid the multiple scattering effects. All measurements were

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done in triplicate at 25°C and the results were expressed as the mean of three measurements.

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2.4 Transmission electron microscopy (TEM) and optical microscopic images

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Images of proteins-polysaccharide interaction were captured with JEOL JEM-2100F (Model EM-

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20014) transmission electron microscope at 7800X. The samples were absorbed on the surface of

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a carbon coated copper grid and fixed with a negative staining method (with 1 % uranyl acetate in

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milli Q water). Due to the high magnification of TEM, the solutions were diluted to 100 times 6 ACS Paragon Plus Environment


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with milli Q water to observe the coacervates while in case of microscopic analysis, the solutions

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were used without any dilution. Furthermore, ALG-proteins images were obtained by using an

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optical microscope (Olympus BX51).

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2.5 Isothermal titration calorimetry (ITC)

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The enthalpic and entropic changes of BLG (0.2%)-ALG (0.1%) and BSA (0.1%)-ALG (0.1%)

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interactions at 25°C was measured using conducted ITC 200 Micro calorimeter (Northampton,

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MA, USA). Separate solutions of BLG, BSA and ALG were made by stirring gently for 8h in 5

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mM sodium citrate buffer (pH 4.2). Sodium citrate buffer was used to eliminate

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protonation/deprotonation effects during the measurement. Sodium citrate buffer was for control

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experiments. Before the measurements, the solutions were degassed for 8 min under vacuum. This

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was done by a device provided by the ITC apparatus. ALG solution was put into the injector

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stirrer syringe (40 µL). Portions of 2 µL (except for the first injection that was 0.4 ml) of ALG

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solution (0.1% (w/w)) were injected sequentially into the titration cell initially containing either

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BLG or BSA solutions or the pure buffer. Each injection took 1sec with extra equilibration time

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between consecutive injections of 150sec

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blank titration of ALG solution into citrate buffer was done and the measured heat was subtracted

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from experiments with proteins present to obtain correct enthalpy change. The change in enthalpy

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per gram ALG (kcal/g), injected into the reaction cell was reported as results. Thermodynamic

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parameters including binding stoichiometry (N), affinity constant (Ka), enthalpy (∆H) and entropy

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(∆S) changes were calculated by iterative curve fitting of the binding isotherms using the one-

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binding-site model (assuming one independent binding site exists for any positively charged

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amino acids) and was plotted against the protein:ALG molar ratio.

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2.6 Statistical analysis

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. The speed of stirrer was set at 750 rpm. First the


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All tests were conducted in triplicates with freshly prepared samples. Analysis of variance

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(ANOVA) was used for data analysis (SPSS 21, Chicago, IL, USA). When F-values were

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significant (P < 0.05) in ANOVA, Duncan’s multiple range test was used to compare treatment

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means.

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3. RESULTS AND DISCUSSIONS

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3.1 pH-dependent complexation of sodium alginate/protein by spectrophotometry

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Turbidimetric measurements have been broadly employed as a practical tool to monitor the

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complex coacervation process in various protein polysaccharide systems

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measurements as a function of pH was performed to evaluate the complexation between ALG

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(0.1% wt) with proteins (BSA and BLG) at different protein concentrations (0.1, 0.15 and 0.2%

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wt). Under the same conditions, homogenous biopolymer solutions of ALG, BSA and BLG

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(0.1%wt) were investigated separately as control samples.

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Turbidity of the solution can be changed by the change in size of aggregates and mass; thus any

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change in turbidity could be caused by formation and dissociation of protein-ALG complexes.

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Accordingly, turbidimetric measurements can demonstrate the complex coacervation process

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between protein and polysaccharide 29.

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ALG solution alone, stayed transparent from pH 7 to 1 Particles formed were not big enough to

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scatter light strongly because of electrostatic repulsion between ALG solution (Figure 1)18.

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Whereas, at lower pH values, there was a minor increase in turbidity, that could be due to

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protonation of ALG carboxyl groups and consequently the degree of the electrical charge on the

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ALG molecules reduced18. The BSA and BLG solutions (without ALG) had a smaller peak,

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individually, at pH ranged about from 4 to 5.2 that was a result of aggregation and the following

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precipitation of BLG and BSA (Figure 1). The electrostatic repulsion between proteins decreases

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around their pI and initiates aggregates pI 30,31. 8 ACS Paragon Plus Environment

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. Turbidimetric


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At pH 7.00, both BLG-ALG and BSA-ALG dispersions were clear, indicating no absorbance at

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400 nm (Figure 1&2). Due to acid titration of BSA-ALG and BLG-ALG dispersions, the four

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phases of turbidity changes were indicated. The presence of ALG to each protein solution, at a 1:1

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protein-ALG mixing ratio caused a significant shift in the initial rise in turbidity from comparing

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to each protein pure solution. The absorbance values were low and constant until pHC. Later on

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the absorbance increased, implying attractive interaction between ALG and proteins 32. At this pH

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the formation of soluble and stable complexes is initiated

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second boundary pH value (pHϕ1), the interaction between the two macromolecules became

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stronger and phase separation occurred. The absorbance value continued to increase to maximum

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value at pHopt. From this phase forward the absorbance values decrease abruptly with lowering pH

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that is due to the sedimentation of particles in protein-ALG dispersions. This was followed by an

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increase in optical density during pH titration and then the curve went down to pHɸ2. Ultimately,

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in very low pH values because of the low charges of ALG chains, as well as the repulsion forces

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between the positively charged proteins, the protein-ALG coacervates could redissolve into

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soluble complexes, or even into non-interacting protein molecules and polysaccharide chains 4.

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When the concentration of protein was increased from 0.1 to 0.15 and 0.2%wt in the solution, a

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slightly higher turbidity curve was found comparing to the lower concentration of protein. It was

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hypothesized that the presence of a highly charged polysaccharide at a concentration of 0.1% (wt)

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caused adequate electrostatic repulsion to hinder protein aggregation at lower protein

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concentration, 0.1% (wt), and small reticence of structure formation can contribute to the minor

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reduction in maximum intensity. When the protein: ALG ratio increased (i.e. protein increased and

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ALG levels decreased), delay of aggregate growth was less substantial where the shifting of pH-

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dependent turbidity profiles to more acidic pH was reduced. With the addition of ALG, the shift to

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lower pH values suggest structures with increased stability at low pH values relative to protein 9 ACS Paragon Plus Environment

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. By decreasing pH passing the

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aggregates in the absence of ALG. Klemmer et al. observed similar trends for mixtures of pea

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protein isolate and alginate polysaccharides with biopolymer ratio of 1:1 to 20:133.

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3.2 Size measurement of protein/sodium alginate

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Particle size analysis is mostly used for monitoring formation and growth of electrostatic

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complexes between protein and polysaccharide. Accordingly, during complex coacervation the

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particle size of BSA-ALG and BLG-ALG was examined to provide further insight into their

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interaction. Protein solutions at all pH values had a particle size ranging from 0.5 to 20 nm (Figure

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2 (c) & (d)). The particle size measurements show that when the pH is reduced to 4.8 the proteins

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tended to form large aggregates. It has been revealed that lowering pH to less than pI of the

205

proteins (4.9 for BSA and 5.2 for BLG) leads to aggregation of proteins 34. Hence the presence of

206

larger particles causing high peaks is negligible

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positive charges on proteins so the proteins exist in the form of individual molecules again as a

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result of repulsion forces. Hence, large aggregates are changed to smaller particles. Therefore, at

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pH 1.8 the particles are smaller for both proteins.

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Three separate peaks were detected in BSA-ALG solution, at pH 4.8 (Figure 2a). The maximum

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particle diameters of 33 nm, 12 nm, and 4 nm, respectively. The 4 nm peak might be assigned to

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BSA molecules, whereas according to ALG size reported by Motwani et al. bigger peak with

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diameters of 33 nm and 12 nm might be from ALG suggesting no or small interactions between

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ALG and BSA at pH 4.8 36. In case of BLG-ALG, at the same pH (4.8), two peaks were found at

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47 nm and 17 nm. It could be due to polymer interaction between ALG and BLG or self-

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association of biopolymers in solution changing the particle (size) distribution of both ALG and

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BLG.37. As the pH decreases to 4.2 the size distribution develops into a more homogenous pattern

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comparing to higher pH values, suggesting the development of complexes between proteins and

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ALG.

8,35

. However, lowering pH develops more



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With more decrease in pH up to 2.8 in BSA-ALG, four different peaks were observed. The

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particle size ranged from o.6 nm to more than 1000 nm but the percentage of smaller particles was

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touched to maximum number (about 8%) at pHopt while in case of BLG-ALG, smaller particles

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reached a maximum number of 22% (at pHopt) as shown in Figure 2a and 2b. This result was in

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accordance with the development of insoluble complexes. With the decrease of pH, the proteins

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and ALG molecules exposed more binding sites for interaction, which generates the development

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of insoluble complexes. At pHɸ2, the insoluble complexes initiated to dissolve and dissociate

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because of advanced protonation of carboxyl groups on the ALG structure 8. In BSA-ALG

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solution at pH 1.8, three peaks were seen. The largest one at around 100nm could be contributed

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to ALG precipitates that occurs in pH values lower than pKa of ALG 38. The smaller peaks appear

230

to be the particles caused by degradation of protein in low pH value. It was suggested that there

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were no interactions between BSA and ALG because of the protonation of anionic groups i.e.

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carboxyl groups on polysaccharide structure thus, similar net charges were carried by both

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biopolymers, at this pH value (Fig. 2a) 8. The rise in the particle size of ALG-proteins mixtures at

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pHɸ2 could be due to the denaturation and unfolding of BSA and BLG at low pH values 39. Liu et

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al. (2015) found the similar results in flaxseed gum and BSA solutions at different pH values (5.6

236

to 2.0) 8.

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3.3 Zeta potential of protein-sodium alginate

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The formation of insoluble and soluble protein-ALG complexes is generally initiated by by the

239

electrostatic attractions between the two biopolymers 6. For a better understanding of electrostatic

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interactions between ALG and proteins in different pH values the electrokinetic potential at the

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slip plane of moving particles, was measured that is zeta potential. The zeta potential of ALG

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solution was negative in entire pH range investigated, which decreased from of -19.05 to -46.24


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(pH from 4.80 to 2.80). The decrease in pH value caused the ionization of carboxylate residues on

244

the ALG molecular structure 40.

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In cases of protein suspensions, as shown in Table 1, during the acid titration, the zeta potential

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value was increased through protonation of carboxyl groups and protein amine 6. The pH, at which

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zeta potential reaches zero, is often considered as isoelectric point (pI). However, the pI of both

248

proteins were slightly lower than what was suggested previously due to the use of minimum

249

concentration of these proteins in the solution and the charge contribution of some impurities in

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proteins

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the reason that proteins were below their pI and accordingly had a net positive charge. Hosseini et

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al. (2013) found similar results by using BLG in higher concentration 35.

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Factors such as polysaccharide type and concentration affect the absolute value of the zeta-. ALG

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has a higher negative charge as compared to many other polysaccharides. It should be noted that

255

the zeta-potential of the mixtures represents the net zeta value of protein-ALG complexes and

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non-interacting individual biopolymers

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showed an increasing trend in general with decreasing pH. Zeta potentials at low pH values

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indicate that the strongest protein-ALG interactions showed in spectrophotometry occurred when

259

the electrical charge of the mixtures was nearly neutralized. Phase separation can appear with a

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negative total charge therefore, at phase separation neutrality is not a requirement and a generic

261

rule for protein-polyanionic polymers 43. Beyond these points, the zeta-potential values remained

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rather constant at limiting values reflecting an excess of polysaccharide. As a result, negatively

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charged polysaccharide molecules associated with positively charged protein and drove charge

264

reversal. The zeta-potential profile of the protein-ALG mixed systems exhibited a similar trend

265

with lower intensity.

41

. The zeta potential of the protein suspension was positive at pHɸ1, pHopt and pHɸ2, for

35,42

. The zeta-potential of BLG-ALG and BSA-ALG also



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Complexation between BSA-ALG and BLG-ALG- at pH 4.8 and 4.2 caused low negative zeta

267

potential values between ~-40 mV - -50 mV (table 1) that makes it a stable system since colloidal

268

particles with zeta potential more than +30 mV and more negative than -30 mV are normally

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considered stable 31.

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3.4 Transmission electron microscopy and light microscopic images

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Optical microscopy was used to study the effect of pH on BSA-ALG and BLG-ALG complexes at

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protein-ALG ratio of 2:1 (w/w) with a total biopolymer concentration of 0.3% (w/w). The images

273

revealed that microstructures of dispersions were different at each critical pH values. At pH 4.8,

274

there were less number of coacervates as shown in Figure 3a, 4e, 5a and 6e while at pH 4.2

275

(Figures 4b, 4f, 5b and 6f), maximum coacervation with minimum particle aggregates was visible.

276

The particle size of coacervates ranged from 150 to 350 nm in both complexes (BSA-ALG and

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BLG-ALG complexes). Gelatin and cashew gum coacervation with a ratio of 1:2.5 had the

278

coacervates about 20 µm at pH 4 to 4.2 44.

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More aggregates were observed at pHopt due to higher turbidity while at pHɸ2, less number of

280

aggregates were visible due to the dissociation of the complexes (Figure 3-6 c, d, g and h). The

281

size of the aggregates in both protein complexes with ALG ranged from 0.20 to 200 µm at pHopt

282

and pHɸ2. These outcomes were also justified by the results of particle size and turbidity

283

measurement.

284

In case of ALG-BSA at pH 4.2, clear black coloured particles with a diameter around 0.4 to 1 µm

285

(Figure 3b) were seen. These particles are expected to be BSA aggregates, due to their irregular

286

shape. On the same image, the smaller white particles were visible with a diameter about 150 nm.

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Due to the round clear structure of these particles, they could be coacervates 45. However, in case

288

of BLG-ALG, at the same pH value, the black aggregates are larger (0.4 to 3 µm) but the

289

coacervates are smaller than BSA-ALG (Figure 3 b) This could be because of the low molecular 13 ACS Paragon Plus Environment

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weight of BLG than BSA

. Considering our results at pH 4.2 for BSA-ALG and BLG-ALG, it

291

could be used for further application such as encapsulation due to maximum coacervate

292

production.

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Figures 4c, d and Figure 6c, d show an increase in particle polydispersity. A large number of

294

BSA-ALG and BLG-ALG particles at pHopt and pHɸ2 displayed larger aggregates than at pHC and

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pHɸ1 indicating the maximum number of complexes at pHopt due to highly charged biopolymers

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and precipitation of biopolymers at pHɸ2. These results are in accordance with our zeta potential

297

results showing a low positive charge for protein-ALG complexes at these two pH values

298

revealing a dispersion with unstable particles and zeta potential lower than +30 mV 31.

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3.5 Isothermal titration calorimetry (ITC)

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Isothermal titration calorimetry is a useful technique to assess energetic and binding parameters in

301

the complex coacervation process by titration of one biopolymer with another in a reaction

302

chamber and measuring the heat absorbed or released

303

resulting from the injections of ALG dispersions into protein (BSA and BLG) solutions at pH 4.2

304

and 25oC are shown in Figure 7 and. 8. In case of both BLG-ALG and BSA-ALG, the area under

305

each peak showed the heat exchange within the cell having BLG after each ALG injection.

306

Initially, a sequence of strong successive exothermic peaks of decreasing intensity was initially

307

observed. With more injections, the released binding energy continuously declined and reached to

308

a state of thermodynamic stability after the 32nd injection for BLG-ALG when a molar ratio of

309

0.75 was reached (Figure 7) and 36th injection for BSA-ALG when a molar ratio of 0.065 was

310

reached (Figure 8).

311

Exothermicity is imparted to nonspecific electrostatic neutralization of opposite charges carried by

312

two biopolymers revealing an enthalpic contribution of complex coacervation

313

decrease in exothermicity could be due to a reduction in free protein molecules remaining in the

18

. The heat flow versus time profiles


47,48

. However, the


314

reaction chamber after injections resulted in reduction in released energy. Hosseini et al. and

315

Hadian et al. reported similar exothermic sequence for BLG interaction with ALG and Persian

316

gum 18,49.

317

Thermodynamic changes during titration were figured out by measuring binding isotherms fitted

318

using a binding model for one binding site and processed by integrating the isotherm peaks and

319

subtraction of heat of dilution of ALG into buffer solution (Figure 7 and 8, lower panel). As a

320

binding site a positively charged amino acid (at the pI) on either BLG or BSA was assumed. The

321

concentration of binding sites was calculated by multiplying the molar concentration of the

322

number of their basic amino acids. Binding stoichiometry (N), affinity constant (Ka), enthalpy

323

(∆H) and entropy contributions (∆S) as thermodynamic parameters were calculated for the

324

interaction of BSA-ALG and BLG-ALG (Table 2).

325

The binding stoichiometry (N) of BLG-ALG was calculated using charge molar ratio to be

326

0.00521 ± 0.0007, suggesting that BLG became saturated with 0.00521 molar charges of ALG

327

while in case of BSA-ALG, 0.00852 molar chargers of ALG were required to bind one molecule

328

of BSA. This equals to the molar ratios of BLG: ALG = 0.784 µM:0.017 µM and BSA: ALG =

329

0.485 µM:0.017 µM.

330

The binding constant (Ka) values point out that the binding of BSA and BLG to ALG can be

331

considered as a high affinity, however in case of BSA it is almost two times bigger than BLG. At

332

pH 4.2, BSA carried highly opposite charge, then more binding sites are available for the BSA-

333

ALG interaction, whereas in BLG-ALG, the Ka value was found to be lower. That shows less

334

number of sites for the interaction of ALG with BLG at the same pH.

335

The binding enthalpies were negative and favourable. Weak complexes are attributed by a

336

negative enthalpy as a result of electrostatic attraction whereas the counter-ion release entropy

337

plays an important role when the interaction became stronger 50. 15 ACS Paragon Plus Environment

Page 16 of 34

Page 17 of 34


338

In soy protein isolate-chitosan coacervation at pH 6.0, the interaction was driven by both enthalpy

339

and entropy, showing that the interaction was a combination of changes in the electrostatic

340

interactions between the charged groups and entropy of the release of counter-ions 51.

341

4.

342

Sodium alginate, an anionic polysaccharide formed complex coacervates with two milk proteins,

343

β-lactoglobulin and bovine serum albumin through electrostatic interactions under specific

344

conditions.

345

Insoluble or soluble complexes between milk proteins ALG can be formed as a function of

346

solution pH and biopolymers mixing ratio. Protein-ALG ratio of 2:1, were selected for further

347

analysis. Formation of soluble coacervates initiated in the range between pHC and pHɸ1 due to

348

binding of anionic ALG to cationic patches on milk protein surfaces. Insoluble complex formation

349

occurred in the range pHɸ1 and pHɸ2 where ALG and proteins were oppositely charged, inducing

350

strong electrostatic attractive interactions.

351

According to ITC results at pH 4.2 for BSA-ALG and BLG-ALG exothermic heat transfer was

352

observed. The binding enthalpies for both systems were negative and favourable. Because of more

353

positive charges on BSA at pH 4.2 the binding constant is almost two times bigger than BLG.

354

Thus, there were more binding sites available for BSA-ALG interaction which is in accordance to

355

zeta potential results at pH 4.2 showing a higher positive charge for BSA comparing to BLG.

356

Results from current research indicated that electrostatic attractive interaction established complex

357

coacervate formation between milk proteins and ALG. This study provides fundamental

358

background knowledge to suggest the implementation of milk proteins-ALG interactions in

359

biomaterial, food, cosmetic and pharmaceutical products.

CONCLUSION

360 361

ACKNOWLEDGMENTS 16 ACS Paragon Plus Environment


362

The authors thank Claudia König for assistance with the TEM measurements. This project was

363

partially financed by the IGS BioNanoTech graduate school (Bundesministerium für Bildung und

364

Forschung (BMBF), Austria).

365 366

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TABLES Table 1. Zeta potential (mV), of ALG 0.1-BSA 0.2 (0.3%), ALG 0.1-BLG 0.2 (0.3%), BSA (0.2%) and BLG (0.2%) solutions at critical pH values pH

sample

BSA-ALG

BLG-ALG

BSA

BLG

ALG

pHC: 4.8

-50.76±1.07 a

-48.77±0.65a

-0.21±0.16a

-1.00±0.52a

-19.05±0.85

pHɸ2: 4.2

-39.66±1.60 b

-39.73±1.07b

11.97±2.84b

7.21±1.15b

-32.10±0.77

pHopt: 2.8

9.73±2.47 c

-

30.63±2.37b

13.57±1.66c

-46.24±2.31

pHopt: 2.4

-

22.43±3.40d

-

-

-

pHɸ1: 1.8

6.97±1.75 c

15.47±1.93c

6.98±1.66b

pHɸ1: 1.6

-

-

-

2.01±0.38c

-

* a–d Means with different letters within each column differed significantly (p < 0.05).

Table 2. Thermodynamic parameters of binding between BSA (0.1%) and BLG (0.2%) with ALG (0.1%) at 25°C in 5 mM sodium citrate buffer (pH 4.20) Parameters

N

Ka (M-1)

∆H (cal/mol)

∆S (cal/mol*°C)

BSA-ALG

0.00852±0.002

7.27*103±1.46*103

-6.076*104±1.78*104

-186

BLG-ALG

0.00521 ± 0.0007

3.99*103± 2.13*103

-9.845*104± 2.124*105

-314

Samples



Figures

3,0 (a)

pH opt

pHopt

Absorbance at 400nm

2,5

2,0

pH2φ2 pH

1,5 pHpH 1

φ1

1,0

pHpH c C

0,5

0,0

0

1

2

3

4

5

pH

6

7

8

0.04 0.03

ALG 0.1 %-BSA 0.1 % ALG 0.1 %-BSA 0.15 % ALG 0.1 %-BSA 0.2 % ALG 0 %-BSA 0.1 % ALG 0.1 %-BSA 0 %

0.02 0.01 0.00 0

2

4

6

3

Absorbance at 400 nm

(b)

pHpH optopt

2

pH φ2

pH 2

pH 1

1

pHφ1 pHpH c C

0

0

1

2

3

4

pH

5

6

7

8

0.02 0.02

ALG 0.1 %-BLG 0.1 % ALG 0.1 %-BLG 0.15 % ALG 0.1 %-BLG 0.2 % ALG 0 %-BLG 0.1 % ALG 0.1 %-BLG 0 %

0.01 0.01 0.00 0

2

4

6

Figure 1. Phase diagram demonstrating critical pH values as a function of pH for BSA-ALG (a) and BLG-ALG (b)


Page 26 of 34

Page 27 of 34


25 (a)

Number (%)

20

15

10

5

0 0,1

1

10

100

1000

10000

1000

10000

LogSize size (d.nm) (d.nm) pH 4.8 pH 4.2 pH 2.8 pH 1.8

25 (b)

Number (%)

20

15

10

5

0 0,1

1

10

100

Log size(d.nm) (d.nm) Size pH 4.8 pH 4.2 pH 2.4 pH 1.6



25 (d)(c) pH 4.8 pH 4.2 pH 2.8 pH 1.8

Number (%)

20

15

10

5

0 0,1

1

10

100

1000

10000

Size (d.nm) Log size (d.nm)

25 (e)(d)

pH 4.8 pH 4.2 pH 2.4 pH 1.6

Number (%)

20

15

10

5

0 0,1

1

10

100

1000

10000

(d.nm) Log sizeSize (d.nm)

Figure 2. Particle size distributions of ALG 0.1 %-BSA 0.2 % (a), ALG 0.1 %-BLG 0.2 % (b) BSA

0.2% (c) and BLG 0.2% (d) solutions at critical pH values


Page 28 of 34

Page 29 of 34


a

b

d

c

Figure 3. TEM images (7800x) of ALG (0.1%)-BSA (0.2%) dispersions at four pH values: 4.8(a), 4.2(b), 2.8(c) and 1.8(d)



e

f

g

h

*Red square shows the coacervates which are spherical particles with clear center

Figure 4. light microscopic images (20X) of ALG (0.1%)-BSA (0.2%) dispersions at four pH values: 4.8(e), 4.2(f), 2.4(g) and 1.6(h)


Page 30 of 34

Page 31 of 34


a

b

c

d

Figure 5. TEM images (7800X) of ALG (0.1%)-BLG (0.2%) dispersions at four pH values: 4.8(a), 4.2(b), 2.4(c) and 1.6(d)



e

f

g

h

*Red square shows the coacervates which are spherical particles with clear center

Figure 6. Light microscopic images (20X) of ALG (0.1%)-BLG (0.2%) dispersions at four pH values: 4.8(e), 4.2(f), 2.4(g) and 1.6(h)


Page 32 of 34

Page 33 of 34


Figure 7. Thermograms (upper panel) and binding isotherms with theoretical fits (lower panel) concerning ALG dispersion (0.1%) in the titration of the BLG dispersion (0.2%), each dissolved in sodium citrate buffer solution (5 mM, pH 4.20), at 25oC)



Figure 8. Thermograms (upper panel) and binding isotherms with theoretical fits (lower panel) concerning ALG dispersion (0.1%) in the titration of the BSA dispersion (0.1%), each dissolved in sodium citrate buffer solution (5 mM, pH 4.20), at 25oC)


Page 34 of 34

Complex Coacervation of Milk Proteins with Sodium Alginate - Journal

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