Nanoencapsulation Systems Based on Milk Proteins and

Chapter 8

Nanoencapsulation Systems Based on Milk Proteins and Phospholipids 1

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Harjinder Singh , Aiqain Ye , and Abby Thompson

Downloaded by UNIV OF SYDNEY on May 3, 2015 | http://pubs.acs.org Publication Date: March 3, 2009 | doi: 10.1021/bk-2009-1007.ch008

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Riddet Centre, Massey University, Private Bag 11 222, Palmerston North, New Zealand Current address: Fonterra Co-operative Limited, Palmerston North, New Zealand 2

Milk contains several components that can be utilized to make nanoparticles for encapsulation and delivery of bioactive compounds. Caseins in milk are essentially natural nanoparticles, designed to deliver essential nutrients, in particular calcium. Similarly, whey proteins, particularly βlactoglobulin, have been designed by nature to bind and transport hydrophobic molecules. The ability of milk proteins to interact strongly with charged polysaccharides opens up further possibilities for making novel hybrid nanoparticles. Phospholipid-rich fractions, extracted from fat globule membranes, can be used to form liposomes. Due to their high sphingomyelin content, these liposomes have some unique stability and entrapment characteristics.

© 2009 American Chemical Society

In Micro/Nanoencapsulation of Active Food Ingredients; Huang, Q., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.

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Since the end of the 20 century, there has been a growing realization of the pivotal link between diet and human health. Consequently, the food industry has created a new category of foods, the so-called functional foods. To fully realize this opportunity the food industry must address several critical challenges, including discovering the potential bioactivity of beneficial compounds, establishing optimal intake levels, and developing adequate food delivering matrix and product formulations. Traditionally, microencapsulation can be used for many applications in the food industry including stabilizing the core material, controlling the oxidative reaction, providing sustained or controlled release, masking flavors, colors or odors, to extend shelf life or protect components against nutritional loss. In recent years, there is considerable interest in developing high performance delivery vehicles for encapsulation and protection of biologically active substances of food origin. Nanosciences, which investigates how to build matter on nanometer scale, usually between 1 and 100 nm, by manipulating individual molecules or atoms, has the potential to provide new solutions in many of these fronts. Certainly nanoparticles may seem attractive as delivery vehicles. By carefully choosing the molecular components, it seems possible to design particles with different surface properties. These nanoparticles are able to encapsulate and deliver the active compounds directly to appropriate sites, maintain their concentration at suitable levels for long periods of time, and prevent their premature degradation. Research efforts are already being made to develop food-based delivery vehicles, such as protein-polysaccharide coacervates, multiple emulsions, liposomes and cochleates. Milk contains several components that can be utilized to make nanoparticles for encapsulation and delivery of bioactive compounds. Caseins in milk are essentially natural nanoparticles, designed to deliver essential nutrients, in particular calcium. Similarly, whey proteins, particularly β-lactoglobulin, have been designed by nature to bind and transport hydrophobic molecules. Milk proteins interact strongly with charged polysaccharides, creating possibilities for novel hybrid nanoparticles. Phospholipid-rich fractions, extracted from fat globule membranes, can be used to form liposomes. These liposomes have some unique stability and entrapment characteristics for both hydrophobic and hydrophilic molecules. This paper provides an overview of potential nanoparticle-based delivery systems, based on milk proteins and phospholipids. A particular focus is placed on recent work in the area of protein-polysaccharide nanoparticles and liposomes carried out in our laboratory at Massey University in New Zealand.

Milk Proteins as Potential Nano-encapsulation Systems Normal bovine milk contains about 3.5% protein which can be separated into caseins and whey proteins (/). Caseins can be fractionated into four distinct


133 proteins, ot , a -, β- and κ- caseins; these represent approximately 38%, 10%, 36% and 12% of whole caseins, respectively. The structures and properties of caseins have been extensively studied (2). In comparison with typical globular proteins, the structures of caseins are quite unique. The most unusual feature is the amphiphilicity of their primary structure. The hydrophobic residues and many of the charged residues, particularly the phosphoserine residues, in the caseins are not uniformly distributed along the polypeptide chain. Therefore, all four caseins have a distinctly amphipathic character with separate hydrophobic and hydrophilic domains, with relatively open and unordered secondary structures. As an example, the distribution of charged residues and hydrophobicity as a function of sequence position of β-casein is shown in Figure 1. β-Casein has two large hydrophobic regions (55-90 and 130-209). The N terminal 21-residue sequence has a net charge of -12, while the rest of the molecule has no net charge. Because the casein monomers cannot sufficiently remove their hydrophobic surfaces from contact with water, the caseins tend to associate with themselves and with each other. In addition, all caseins are able to bind calcium with the extent of binding being proportional to the number of phosphoserine residues in the molecule. a and a -caseins are most sensitive to calcium followed by βcasein while κ-casein is insensitive to calcium. κ-Casein is capable of stabilising other caseins against calcium-induced precipitation and allows the formation of colloidal size aggregates. The unique physio-chemical properties of caseins have been traditionally exploited to modify and enhance textural and sensory characteristics of foods (J). Casein and caseinates can bind water, stabilise foams, emulsify fat and control viscosity in formulated foods, in addition to providing high nutritional value. Caseins also possess many of the properties required of a good wall material for encapsulation (4). The ability of caseins to self-assemble into particles of varying sizes with different stability characteristics offers opportunities for nanoencapsulation for the delivery of bioactive compounds. A recent study (5) showed that hydrophobic compounds, such as vitamin D2, can be incorporated into casein particles, formed by the re-assembly of caseins. These reassembled casein particles can provide partial protection against UVlight-induced degradation of vitamin D2 entrapped in them. Further understanding of "surfactant-like" self-assembly properties of caseins would allow us to create novel nanometer-scale structures suitable for delivery of bioactive compounds. The principal fractions of whey proteins are β-lactoglobulin, bovine serum albumin, ct-lactalbumin and immunoglobulins (I). In contrast to caseins, the whey proteins possess high levels of secondary, tertiary and, in most cases, quarternary structures. β-Lg is built up of two β-sheets, formed from nine strands converging at one end to form a hydrophobic calyx or pocket, and a flanking three-turn α-helix (6). This pocket serves as a binding locus for apolar


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Figure 1. Distribution of charged residues and hydrophobicity as a function of sequence position ofβ-casein.


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135 molecules such as retinol (7) and long-chain fatty acids (8). Similarly, bovine serum albumin binds a large variety of compounds, including retinol and longchain fatty acids (P). The concept of using ligand-selective whey proteins for delivery and protection of active agents is relatively new, and such systems need further development. The ability of whey proteins to aggregate and form gels during heat treatment is of considerable importance (1,3). By controlling the assembly of protein molecules during the aggregation process, hydrogels, micro- and nanoparticles suitable for the delivery of bioactive compounds can be produced. For example, a monodisperse dispersion of 40 nm whey protein nanospheres was obtained by Chen et al. (10) by heating whey proteins at relatively low protein concentration and ionic strength and a temperature around 55°C. The potential of these nanospheres as carriers of nutraceutical agents was studied in vitro; it appears that protein nanoparticles could be internalized by cells and degraded therein to release nutraceutical compounds. Partial hydrolysis of whey protein, α-lactalbumin, by a protease from Bacillus lichenifromis has been shown to produce peptides that self-assemble into nanometer-sized tubular structures under certain conditions (11). These micrometer long hollow tubes, with a diameter of only 20 nm, have potential applications in the delivery of nutraceuticals.

Milk Proteins-Polysaccharide Composite Systems Protein structure can be modified through processing treatments or change of solution conditions to allow formation of complexes with polysaccharides. A wide variety of nutrients can be incorporated into these complexes by relatively non-specific means. Specific binding of a nutrient to amino acid side chains can also be achieved in some cases. At pH values below their isoelectric points (pi), proteins carry positive charges and can interact with polysaccharides bearing carboxylic, phosphate, or sulfate groups. This inter-biopolymer complexation of positively charged proteins and anionic polysaccharides can lead to the formation of soluble and insoluble complexes (12). Interbiopolymer complexes can be regarded as a new type of food biopolymer whose functional properties differ strongly from those of the macromolecular reactants. The complex coacervation of globular proteins and polyelectrolytes, e.g., gelatin, β-lactoglobulin, bovine serum albumin, egg albumin, and soy protein, has been extensively studied (13-14). Recently, we have observed the formation of soluble and stable complexes on the mixing of gum arabic and sodium caseinate at a wide pH range (pH 4 to 5.4); the complexes resulted in stable dispersions with particle size between 100 to 200 nm (15).


136 Mixtures containing 0.1% sodium caseinate and 0.5% gum arabic were acidified by dropwise addition of HC1 from pH 7.0 to pH 2.0 and samples were stored for 24 h at 20°C. The absorbance profiles of mixtures are shown in Figure 2. The absorbance values of 0.1% sodium caseinate solution abruptly increased at pH 5.4 and reached a maximum at pH 5.0 (Figure 2). Further decrease in pH caused a decrease in absorbance due to large-scale aggregation and subsequent precipitation of the caseins around their pi. In contrast, the absorbance of sodium caseinate/gum arabic mixtures increased slightly at pH 5.4 (pH ) but remained almost constant between pH 5.4 and pH 3.0. No phase separation occurred in this pH range (see photographs of 0.1% sodium caseinate/0.5% gum arabic mixtures at different pH values in Figure 2). On decreasing the pH below pH 3.0, the absorbance values increased and phase separation took place subsequently. At pH 2.0, the absorbance values of the sodium caseinate/gum arabic mixture decreased. Particle sizes of these stable dispersions in the pH range from 5.4 to pH 3.0, measured using dynamic light scattering, showed that the average diameter of the particles in the sodium caseinate/gum arabic mixtures remained stable at approximately 110 nm as the pH was decreased from pH 5.4 to pH 3.0 and increased to very large values (> 10 μπι) when the pH was reduced further. Electron microscopy confirmed the presence of these composite nanoparticles between pH 5.4 to pH 3.0 (15). The mechanism of the formation of these nanoparticles based around the self-aggregation of casein and the electrostatic interaction between the aggregated particles of casein and gum arabic molecules has been proposed (15, Figure 2). As the pH of the mixture decreases below pH 5.4, the caseinate molecules tend to undergo small-scale aggregation prior to large-scale aggregation and precipitation at pH values closer to their pi (pH 4.6). In this case, the gum arabic molecules may attach to the outside of these small-scale aggregates in the early stages of aggregation through electrostatic interactions between negatively charged gum arabic and exposed positive patches on the surface of the caseinate aggregates. The presence of hydrophilic gum arabic molecules on the outside of the caseinate aggregate may be enough to sterically stabilise these nano-particles and consequently prevent self-aggregation. As the charge on the nano-particles is quite low, for example, ~15mV at pH 4.0, steric stabilisation is probably important.


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Effect of NaCI and CaCI on the properties of nanoparticles 2

Different amounts of NaCI or CaCl were added to the stable nanoparticle dispersion of caseinate/gum arabic at pH 4.6. The changes in the absorbance and average particle size of mixtures, which were measured after storage for 24 h, are shown in Figure 3. The absorbance value of the dispersions increased with 2



137 increasing NaCI concentration upon to 45 mM but decreased abruptly at higher NaCI concentrations (Figure 3A). The average particle size of particles increased from -105 nm to -450 nm at 40 mM added NaCI. It also was noted that the dispersions containing 0 to 40 mM added NaCI were stable (no phase separation) after 7 days, whereas precipitation occurred at added NaCI > 40 mM (Figure 3 A). The analysis of these precipitated samples showed that there was no protein and nearly 100% of gum arabic remained at the top clear layer (data not shown). This indicated that the precipitation was due to self-association of caseinate molecules, and the association of gum arabic with the casein aggregates was prevented by the added NaCI. This is consistent with our previous work (15) that showed that the complexes can not be formed between the sodium caseinate and gum arabic in the presence of NaCI > 50 mM. At added NaCI < 50 mM, the size of stable complex particles increased with increasing the concentration of NaCI. This could be due to the dissociation of some of the gum arabic molecules from the casein aggregate surface. The amount of gum Arabic remaining is probably sufficient to prevent large scale aggregation and precipitation of casein. The caseinate-gum arabic mixture began to precipitate when the concentration of added CaCl were higher than 3 mM (Figure 3B). At CaCl added < 3mM, the absorbance the nanoparticle dispersions increased with increasing the concentration of added CaCl . The average particle size increased from -105 nm to -220 nm at 3 mM added CaCl . The behaviour of dispersions after addition of CaCl was similar to that after addition of NaCI. This indicated that the effect of added CaCl was because of change in the ionic strength of system. In addition, It was ion bridging that could take place between the divalent calcium ion and the negatively charged casein molecules in the complexes. This may result in an increase in the size of complex particles and precipitation above certain CaCl concentration. 2

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Milk Phospholipids as Potential Nanoencapsulation Systems 1

Milk contains 0.13-0.34 mg mg" protein phospholipids of which about 60% are associated with milk fat globule membrane (MFGM) (16). The most abundant phospholipids are phosphatidylcholine (PC), phosphotidylethanolamine (PE) and sphingomyelin, while phosphatidylserine (PS) and phosphatidylinositol (PI) are present in low amounts (16-17). The MGFM phospholipids contain high levels of long chain fatty acids, such as palmitic (16:0), stéarate (18:0), tricosanoate (23:0) while the short- and medium-chain fatty acids are present in very low levels. The composition of the MFGM phospholipid is very different from the commonly used soy- or egg-derived phospholipids (16). These differences would be expected to influence the selfassembly characteristics of phospholipids produced from the MFGM. It should



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Figure 2. Absorbance as a function of pH off). 1% sodium caseinate solution (O) and mixtures ( · ) of 0.1% sodium caseinate and 0.5% gum arabic at 20 Χ. The mixtures were acidified using HCl and then stored at 4X1for 24 h. The proposed model of Ye et al (15) for the formation of nanoparticles is also depicted. Pictures of 0.1% sodium caseinate and 0.5% gum arabic mixtures at different pH values are also shown.



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Nanoencapsulation Systems Based on Milk Proteins and

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