Formation of protein nanoparticles by controlled heat treatment of lactoferrin: Factors affecting particle characteristics

Food Hydrocolloids 25 (2011) 1354e1360

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Formation of protein nanoparticles by controlled heat treatment of lactoferrin: Factors affecting particle characteristics Carlos Bengoechea b, *, Irene Peinado c, David Julian McClements a a

Biopolymers and Colloids Research Laboratory, Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA Departamento de Ingeniería Química, Universidad de Sevilla, Facultad de Química, Profesor García González 1, 41012 Sevilla, Spain c Institute of Food Engineering for Development, Polytechnic University of Valencia, Camino de Vera s/n, 46022 Valencia, Spain b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 September 2010 Accepted 24 December 2010

Lactoferrin is a globular protein from milk that has considerable potential as a functional ingredient in food, cosmetic and pharmaceutical applications. In this study, we examined the possibility of preparing food-grade bovine lactoferrin (bLf) nanoparticles using a simple thermal processing method. Differential Scanning Calorimetry (DSC), light scattering, and z-potential techniques were used to provide information about the conformational changes, aggregation, and electrical charge of bLf in aqueous solutions. DSC studies indicated that the protein had two thermal denaturation temperatures (61 and 93
C), which were associated with two different lobes of the protein. Protein denaturation was found to be irreversible, which was attributed to the formation of protein nanoparticles, whose size depended on the temperature and duration of the thermal treatment. Higher holding temperatures produced faster protein aggregation and larger protein nanoparticles: 85 > 80 > 75 > 70
C. The protein nanoparticles produced by thermal treatment were resistant to subsequent changes in pH (from 3 to 11) and to salt addition (0e200 mM NaCl). The lactoferrin nanoparticles produced in this study may be useful as function ingredients in commercial products. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Lactoferrin Nanoparticles DSC z-Potential Aggregation Denaturation

1. Introduction Bovine lactoferrin, bLf, is a single-chain glycoprotein with a reported molecular weight around 80 kDa and approximately 700 amino acid residues (Levay & Viljoen, 1995; Öztas¸, Yes¸im, & Özgünes¸, 2005). Lactoferrin is an iron transporter and may be found in both the iron-saturated (holo-lactoferrin) and the irondepleted (apo-lactoferrin) forms. Its structure consists of two lobes with a reversible iron-binding capacity of two atoms per lactoferrin molecule (one for each lobe), with one HCO 3 ion being synergistically bound with each Fe3þ ion. One of the main interests in lactoferrin resides in its various biological properties, such as antimicrobial, anti-inflammatory, anti-tumor, immunemodulatory, and enzymatic activities (Brock, 2002). It seems that when iron is bound into the lactoferrin molecule, a more closed conformation is adopted (Levay & Viljoen, 1995). It has been extensively reported that the isoelectric point (pI) of lactoferrin is around 8e9 (Brock, 2002; Levay & Viljoen, 1995; Moguilevsky, Retegui, & Masson, 1985). This relatively high pI is explained by

* Corresponding author. Tel.: þ34 954557179; fax: þ34 954556447. E-mail address: [email protected] (C. Bengoechea). 0268-005X/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2010.12.014

a unique basic region in the N terminal region of lactoferrin, which can bind acidic molecules (Brock, 2002). There has been considerable interest in the formation of biopolymer particles from proteins and/or polysaccharides (Benichou, Aserin, & Garti, 2002; McClements, 2006; Tolstoguzov, 2002, 2003; Turgeon, Schmitt, & Sanchez, 2007), as they may be used for protection and delivery of bioactive compounds in food systems (Chen, Remondetto, & Subirade, 2006; Emerich & Thanos, 2007; Goldberg, Langer, & Jia, 2007; Ubbink & Krueger, 2006). Biopolymer particles may be formed through complexation of proteins, polysaccharides and other compounds. Alternatively, recent studies have shown that protein nanoparticles and microparticles can be formed by controlled thermal denaturation of globular protein solutions (Bromley, Krebs, & Donald, 2006; Donato, Schmitt, Bovetto, & Rouvet, 2009; Hoffmann, Roefs, Verheul, VanMil, & DeKruif, 1996; Jones, Decker, & McClements, 2010; Santipanichwong, Suphantharika, Weiss, & McClements, 2008). Some authors have combined both approaches to form nanoparticles through heat denaturation of globular proteins followed by electrostatic complexation with polysaccharides (Hong & McClements, 2007; Jones, Lesmes, Dubin, & McClements, 2010; Kelly, Gudo, Mitchell, & Harding, 1994; Sanchez & Paquin, 1997; Schmitt, Sanchez, Desobry-Banon, & Hardy, 1998; Yu, Hu, Pan,

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Yao, & Jiang, 2006). Previous studies have primarily been carried out using b-lactoglobulin to form the nanoparticles, which has an isoelectric point of around pH 5 and a single thermal denaturation temperature. In this study, we examined whether protein nanoparticles could also be formed using lactoferrin, which has a much higher isoelectric point than b-lactoglobulin (and is therefore positively charged over a wider pH range) and has two thermal denaturation temperatures. The stability of lactoferrin particles formed through heating at different pH and ionic strength values will be evaluated. It should be noted, that Sreedhara, et al. (2010) recently studied the pH-induced changes in the structure of a bovine lactoferrin similar to the one used in this work, finding an unfolded structure for the protein under acidic conditions, and folded at higher pH values. Lactoferrin nanoparticles may be useful functional ingredients in food, cosmetic and pharmaceutical applications, e.g., to modify the optical or rheological properties of products, or to encapsulate and deliver bioactive ingredients. Our purpose was to demonstrate that protein nanoparticles could be formed from lactoferrin, and to highlight some of the factors that influence their properties. Further work would be required to translate this approach into an industrial method of producing lactoferrin nanoparticles.

Decker, and McClements (2009) previously used this procedure to create protein-based particulate-suspensions. Following thermal treatments, solutions were removed from the water bath and allowed to cool in a water bath at room temperature.

2. Experimental

2.6. Particle characterization

2.1. Materials

2.6.1. Turbidity measurements The turbidity of 0.2% lactoferrin solutions was analyzed using a UV/visible spectrophotometer at 600 nm (Ultraspec 3000 pro, Biochrom Ltd., Cambridge, UK). Samples measured at room temperature were analyzed in disposable plastic cuvettes with a cell path length of 1.0 cm. Solutions were vortexed for 2e3 s prior to measurement to ensure the samples were homogeneous. Distilled water was used as a blank reference. The change in turbidity with time at various holding high temperatures (75, 80, 85, 90
C) and the change in turbidity with temperature were also analyzed using the same UV/visible spectrophotometer. In this case, samples were placed in cuvettes composed of quartz with a path length of 1.0 cm. Sample solutions were placed into the cuvettes, followed by 3 drops of hexadecane to prevent excessive evaporation. Distilled water was used as a blank reference. In the temperature scanning measurements, the turbidity was measured as the sample temperature was increased from 25 to 95
C at a rate of 0.6
C per minute. Certain samples were also scanned during cooling (95e25
C) at a rate of 0.8
C per minute to determine whether the transitions observed were reversible.

Purified lactoferrin powder (Lot 10410479) obtained from cow’s milk was kindly donated by DMV International (BA Veghel, The Netherlands). The reported composition expressed as a dry weight percent was: 97.7% protein; and 0.12% ash; the remaining water content expressed on the wet basis of the powder was 2.8%. The manufacturer reported that the iron content of the lactoferrin powder was reported to be less than 120 ppm (0.012 wt%), which corresponds to a molar ratio of iron-to-protein of <0.18. It was used directly from the sample container without further purification. All solutions were prepared with double-distilled/de-ionized water obtained from a filtration unit on-site. 2.2. Biopolymer solution preparation Protein solutions were prepared by adding water to a weighed amount of lactoferrin powder and then stirring constantly at ambient temperature for 1 h. Protein solutions were initially adjusted to pH 7.0, which is the most stable form of lactoferrin (Brock, 1997), using a 0.1 N sodium hydroxide solution. Lactoferrin concentrations are reported as weight percentages (wt%) based on the initial masses of the powders. A fixed protein concentration of 0.2 wt% was used in these experiments so as to form solutions that gave a convenient signal in the light scattering and z-potential instrument. Biopolymer solutions were adjusted to pH values below 7.0 using 1.0, 0.1 and/or 0.01 N hydrochloric acid solutions. All solutions were equilibrated at the desired pH for 1e3 min before sample aliquots were removed. All solutions were prepared with double-distilled/de-ionized water obtained from a filtration unit on-site. 2.3. Creation of protein particles through thermal treatment The formation of biopolymer particles by thermal treatment was carried out by first adjusting 0.2% lactoferrin solutions to pH 7, then pouring them into glass test tubes with plastic screw-caps, and then subjecting them to a heat treatment by placing the test tubes in a temperature-controlled water bath at different temperatures (70e90
C) for different holding times (1e60 min). Jones,

2.4. pH stability of protein particles The effect of pH on the properties of the protein particles prepared by thermal treatment (75
C for 20 min) was evaluated using two different approaches: (i) modifying the pH of the solutions (from 2 to 11) before thermal treatment; (ii) modifying the pH of the solutions (from 2 to 11) after thermal treatment. Measurements were performed 24 h after sample preparation. 2.5. Salt concentration The effect of salt on the stability of the protein particles prepared by thermal treatment (75
C for 20 min) was also evaluated using two different approaches: (i) adding 0e200 mM NaCl before thermal treatment; (ii) adding 0e200 mM NaCl after thermal treatment. Measurements when performed 24 h after sample preparation.

2.6.2. Particle size and charge measurements Particle sizes and charges of biopolymer particles were determined using a commercial dynamic light scattering and micro-electrophoresis device (Nano-ZS, Malvern Instruments, Worcestershire, UK). The particle size data is reported as the Z-average mean diameter, while the particle charge data is reported as the x-potential (Jones & McClements, 2008; Jones et al., 2009). 2.6.3. Differential scanning calorimetry Absorption or release of thermal energy from samples was monitored using a differential scanning calorimeter (VP-DSC; MicroCal, Northampton, MA). Sample solutions were degassed in a small degassing unit for 15e20 min at room temperature. Degassed samples and distilled water as a blank were injected into the sample and reference cells of the DSC, respectively, and then temperature scans were carried out from 25 to 100
C at a rate of 1.0
C per minute.

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2.6.4. Statistical analysis All measurements were performed on at least three freshly prepared samples and are reported as means and standard deviations. 3. Results and discussion 3.1. Characterization of thermal denaturation and aggregation of lactoferrin The measured DSC thermogram of a native bovine lactoferrin solution (0.2 wt% bLf, pH 7) is shown in Fig. 1a. Two endothermic peaks were observed at 60.4 and 89.1
C, which correspond to the two denaturation temperatures (Tm) for the same protein reported by previous authors (Mata, Sánchez, Headon, & Calvo, 1998; Paulsson, Svensson, Kishore, & Naidu, 1993; Rüegg, Moor, & Blanc, 1977). These peaks have previously been attributed to differences in the heat sensitivities of the two lobes of lactoferrin (Evans & Williams, 1978), since the C lobe appears more compact than the N lobe in the iron-saturated protein (Anderson, Baker, Norris, Rice, & Baker, 1989), and it has also been explained by the presence of monoferric species (Rüegg et al., 1977). We also examined the thermal behavior of bLf solutions that had been pre-heated prior to DSC analysis. Only a single peak was observed at around 90
C for a bLf solution that had been pre-heated at 75
C for 20 min. This temperature is between the two thermal denaturation peaks observed for the native bLf (Fig. 1a), which suggests that the lobe with the lower Tm had unfolded, but that the lobe with the higher Tm remained intact. No peaks were observed for a bLf solution that had been pre-heated at 91
C for 20 min (Fig. 1a). This temperature

A

bLF unheated 90

bLF 91 ºC

bLF 75 ºC

60.4 ºC

80

Cp (kcal /mol/oC)

70

89.1 ºC

60 50 40

3.2. Influence of holding temperature on protein aggregation

30 20 10 0 30

50

70

90

Temperature (oC)

B

2.0 1.8 1.6

Turbidity (cm-1)

exceeds both thermal denaturation peaks of the native bLf (Fig. 1a), which suggested that both lobes had been irreversibly denatured by this heat treatment. Previous researchers have reported similar effects of pre-heating on the DSC thermograms of bLf; pre-heating at 70 and 80
C reduced the intensity of the first peak, whereas the second peak remained unaltered (Paulsson et al., 1993). It has been suggested that the peaks at 60 and 90
C correspond to the apo- and holo- forms of bLf, respectively (Kussendrager, 1994; Lin, Mason, Woodworth, & Brandts, 1994; Paulsson et al., 1993), implying that the source of lactoferrin used in the present study is a mixture of these two forms. Brisson, Britten, and Pouliot (2007) considered the partly iron-saturated native-Lf as a mixture composed of apo-Lf and holo-Lf, but also possibly of monoferric Lf species saturated at either their N or C lobe. In future studies it would be useful to accurately determine the fraction of native protein in the lactoferrin powder. The turbidity versus temperature profile of the native bLf solutions was measured to obtain information about protein aggregation during heating (Fig. 1b). The turbidity remained relatively constant and close to zero when the samples were heated from 25 to 60
C. A pronounced increase in turbidity occurred from 60 to 65
C, with a maximum slope at 62.3
C (determined from the inflexion point), and then the turbidity remained relatively constant from 65 to 80
C. A steep increase in turbidity occurred from 80 to 90
C with a maximum slope around 88.4
C. The temperature values where the maximum slope of turbidity versus temperature occurred (62.3 and 88.4
C, Fig. 1b) were fairly close to the thermal denaturation temperatures observed in the DSC thermograms (60.4 and 89.1
C, Fig. 1a). We hypothesize that the observed increases in turbidity correspond to protein aggregation induced by thermal denaturation of the globular proteins. When the proteins unfold they expose nonpolar groups normally buried in their non-polar interior, which leads to aggregation through hydrophobic attraction. The DSC and turbidity results suggest that this is a two-stage process. There is limited protein aggregation after the first lobe (lower Tm) of the bLf unfolds, and then more extensive protein aggregation after the second lobe (higher Tm) unfolds.

1.4

88.4 ºC

1.2 1.0 0.8 0.6

62.3 ºC

0.4 0.2 0.0 20

40

60

80

100

Temperature (oC) Fig. 1. (a) Differential scanning calorimetry data for a 0.2% lactoferrin solution (pH 7) unheated (,), heated at 75
C for 20 min (D) and heated at 91
C for 20 min (B). The y-axis has been shifted between samples to improve clarity, but the scale remains; (b) Turbidity (at 600 nm) versus temperature profiles for a 0.2% lactoferrin solution (pH 7) during heating and cooling.

The isothermal aggregation of the globular proteins near their thermal denaturation temperatures was examined by measuring the turbidity increase of native protein solutions (0.2% bLf, pH 7) stored at various holding temperatures (Fig. 2a). In addition, an aggregation time (tA¼1), as the time taken for the turbidity to first exceed a value of 1 cm1 was defined (Fig. 2b). These isothermal aggregation experiments indicated that: (i) higher turbidity was achieved at shorter times when increasing the holding temperature, i.e., shorter tA¼1; (ii) aggregation appeared to be a two-step process. For all samples, there was a relatively rapid initial increase in turbidity over the first five to ten minutes, followed by a period when the turbidity remained relatively constant, followed by another steep increase in turbidity at longer times. The period before which this second rapid increase in turbidity occurred decreased as the holding temperature increased (Fig. 2a). We postulate that the first steep increase in turbidity corresponds to the unfolding of the first protein lobe leading to some protein aggregation, whereas the second increase in turbidity corresponds to the unfolding of the second protein lobe leading to more extensive protein aggregation. Previously is has been reported that the thermal denaturation of bovine lactoferrin between 72 and 85
C followed first-order reaction kinetics (Sánchez et al., 1992). The fact that we observed a two-step process in the turbidityetemperature profiles in this study suggests that protein aggregation could not be described by first-order reaction kinetics.

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A

A

2

1357

150

Z-Average Diameter (nm)

140

Turbidity (cm-1)

1.5

1

75 80

0.5

85

130 120 110 100 90

90 0 0

10

20

30

40

50

60

70

80

80

90

65

70

Holding Time (min)

B

B

90 80

80

85

90

0.35 0.3

70 60

0.25

Turbidity (cm-1)

tA = 1 (min)

75

Holding temperature (ºC)

50 40 30 20

0.2 0.15 0.1

10 0 70

75

80

85

90

0.05

Holding Temperature (oC)

70

75 80

85ºC

0 Fig. 2. (a) Effect of holding temperature on turbidity along time; and (b) time necessary to achieve a turbidity value equal to 1 versus holding temperature for a 0.2% lactoferrin solution (pH 7).

The effect of holding temperature on the characteristics of protein particles formed by thermal treatment was also assessed by holding native protein solutions at temperatures ranging from 70 to 85
C for 20 min. After cooling to ambient temperature, the samples were analyzed by dynamic light scattering and spectrophotometry to determine their particle size and turbidity (Fig. 3). The measured particle size and solution turbidity varied appreciably from batchto-batch after heating (as demonstrated by the large error bars), which suggested that the particle formation process was highly sensitive to initial conditions. Nevertheless, our measurements indicated that the mean particle diameter and/or concentration increased with increasing holding temperature (Fig. 3a and b). This effect can be attributed to the fact that there are more reactive (unfolded) globular protein molecules at higher temperatures, and that the proteineprotein collision frequency will also increase with temperature. 3.3. Influence of holding time on protein aggregation The effect of holding time on the properties of nanoparticles particles formed by heating native protein solutions (0.2% bLf, pH 7) was also examined. The protein solution was held at 75
C for different times ranging from 1 to 60 min (Fig. 4). The mean particle diameter and solution turbidity increased rapidly during the first 5 min of storage indicating that rapid protein aggregation was occurring during this period. The particle size and turbidity then remained relatively constant from 5 to 30 min, suggesting that there was no further change in particle characteristics. These experiments are useful for determining optimum processing

65

70

75

80

85

90

Holding temperature (ºC) Fig. 3. Effect of holding temperature (70, 75, 80, 85
C) on (a) the Z-average mean diameter and (b) turbidity of a 0.2% lactoferrin solution heated for 20 min. A photograph of these solutions is displayed in the bottom graph.

conditions required to form bLf nanoparticles by thermal processing. If the time is too short, then the protein aggregation process will not have fully occurred. On the other hand, if the time is too long, then there will be associated energy and time costs. 3.4. Influence of pH and ionic strength on protein particle stability Functional protein nanoparticles may be exposed to a variety of solutions conditions when they are incorporated into commercial products, such as foods, cosmetics, personal care products, and pharmaceuticals. It is therefore important to establish the influence of various environmental conditions on their stability and functional properties. In this section, we examine the influence of pH and ionic strength on the properties of bLf nanoparticles formed by thermal treatment. The pH and ionic strength of the aqueous solutions were adjusted either before or after the protein nanoparticles were formed by heating (75
C for 20 min) aqueous solutions of native protein (0.2% bLf). The temperature was selected to be intermediate between the first and the second thermal denaturation temperatures, as extensive protein aggregation and sedimentation occur when Lf is heated above the second Tm, but not when it is heated above the first Tm. There were appreciable differences in the influence of pH on the particle properties, depending on whether it was adjusted before or after thermal treatment (Fig. 5). The z-potential versus pH profile of the protein particles was fairly similar for samples prepared by

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C. Bengoechea et al. / Food Hydrocolloids 25 (2011) 1354e1360

A

A

140

Z-Average Diameter (nm)

120

100

80

60

40

75 ºC 20 0

10

20

30

40

50

60

Holding time (min)

B

B

0.3

75 ºC

Turbidity (cm-1)

0.25

0.2

0.15

0.1

C

0.05

0 0

10

20

30

40

50

60

Holding time (min) Fig. 4. Effect of holding time on (a) the Z-average mean diameter and (b) turbidity for a 0.2% lactoferrin solution heated at pH 7 and at a holding temperature of 75
C for 0, 1, 3, 5, 30 and 60 min. A photograph of these solutions is displayed in the bottom graph.

adjusting pH before or after heating (Fig. 5a). The electrical charge on the particles went from positive at low pH to negative at high pH, with a point of zero charge around pH 9, which is in agreement with reported values for the isoelectric point of bLf (Brock, 2002; Levay & Viljoen, 1995; Moguilevsky et al., 1985). On the other hand, the particle size and solution turbidity were highly dependent on when the pH was adjusted relative to thermal treatment. Protein particles prepared by heating native protein solutions at pH 7, and then adjusting them to different pH values afterward, had relatively small diameters (d < 150 nm) and solution turbidities (s < 0.1 cm1) across the entire pH range studied (2e12). However, there was some evidence of increased protein aggregation around pH 9 as indicated by an increase in solution turbidity (Fig. 5b). This effect was attributed to the fact that the protein particles had a low net charge around their isoelectric point, and so there would only be a weak electrostatic repulsion between them. Interestingly, these protein particles did not dissociate when the pH was adjusted far from their isoelectric point, which might have been expected due to the increase in electrostatic repulsion between the individual protein molecules. The good pH stability of the protein particles may be due to the formation of strong irreversible hydrophobic or covalent bonds between the lactoferrin molecules after the protein molecules unfolded (Steijns & Van Hooijdonk, 2000). Visual observation of samples confirmed the turbidity measurements: samples in which the pH was changed after thermal treatment (20 min, 75
C) of a 0.2% bLf solution at pH 7 remained stable and transparent across the whole pH range studied (from 3 to 11), but when 0.2% bLf samples were first adjusted to the

Fig. 5. Effect of pH adjustment prior (C) and after (B) heat treatment on (a) zpotential, (b) turbidity, and (c) mean particle diameter for a 0.2% lactoferrin solution heated at 75
C for 20 min.

required pH and then heated, they formed turbid colloidal suspensions at pH values from 7 to 9. Nevertheless, further research is needed to identify the relative importance of different molecular interactions in order to support this hypothesis. When native bLf solutions were heated after they had been adjusted to a particular pH (from 2 to 12), there was evidence of extensive protein aggregation (higher turbidity and larger particle size) near the protein isoelectric point (Fig. 5b and c). However, protein aggregation was much less at pH values well below (pH  6) or well above (pH  10) the isoelectric point. Abe et al. (1991) also reported little aggregation of bLf solutions at acidic pH when heating at temperatures ranging from 80 to 120
C for 5 min, but an increase in turbidity and sometimes gelation were observed upon heating at neutral and alkaline pH. When the protein solutions were heated at a pH well away from the pI there would be a relatively strong electrostatic repulsion between the individual bLf molecules, which may have limited the formation of large protein aggregates. On the other hand, when the protein molecules were heated at a pH close to their pI, there would only be a weak electrostatic repulsion between them that may have led to

C. Bengoechea et al. / Food Hydrocolloids 25 (2011) 1354e1360

more extensive protein aggregation and the formation of larger protein particles. These studies indicate that the size of the protein particles can be manipulated by controlling solution pH and the order of thermal treatment relative to pH adjustment. The ionic strength of the aqueous solution impacts the magnitude and range of any electrostatic interactions in the system, and therefore should affect the formation and properties of the protein particles formed by heating. The influence of ionic strength on particle formation was studied by adding different amounts of salt (10e200 mM NaCl) to the protein solutions (0.2% bLf, pH 7) either before or after thermal treatment (75
C, 20 min) (Fig. 6). When the salt was added after the protein particles had been formed, the resulting colloidal dispersions remained relatively stable to particle aggregation and sedimentation, having a uniform cloudy appearance (Fig. 6a). On the other hand, when the salt was added before thermal treatment there was evidence of extensive protein aggregation and sedimentation in all of the samples (Fig. 6b). These results can be attributed to the influence of ionic strength on the electrostatic interactions in the system. When salt is added prior to heating, the electrostatic repulsion between the individual proteins is reduced, which promotes the formation of large particles. Kawakami, Tanaka, Tatsumi, and Dosako (1992) also showed that Lf aggregation increases with ionic strength. On the other hand, when the proteins are heated in the absence of salt, there is a strong electrostatic repulsion between them, which limits more extensive protein aggregation. Presumably, any non-polar groups exposed on the bLf surfaces during heating associate with other exposed nonpolar groups, which means that the surface hydrophobicity of the protein particles formed after heating is relatively low. In addition, lactoferrin is known to have some glycosidic residues, which may protrude into the aqueous phase and increase the steric repulsion

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between the protein particles. Brisson et al. (2007) suggested that Lf heat-induced aggregation proceeded via a combination of noncovalent interactions and intermolecular thiol/disulphide reactions that did not require free thiol residues. These effects may account for their relatively good stability to changes in pH and salt after formation. 4. Concluding remarks This study has shown that bovine lactoferrin undergoes a twostep aggregation process upon heating, which was attributed to a two-step protein unfolding process: the thermal denaturation temperatures of the two lobes of bLf were found to be around 60 and 90
C. The size of the particles formed during the corresponding thermal treatment depended on the temperature/time of the heating process. Higher temperatures resulted in a faster aggregation process and the formation of larger protein particles. The particles formed by thermal treatment had good resistance to changes in the pH and ionic strength of the surrounding aqueous phase. The protein nanoparticles produced using the controlled thermal processing approach described in this study may be useful as function ingredients in commercial products, such as foods, cosmetics and pharmaceuticals. For example, they could be used to modify the optical or rheological properties of products, or to encapsulate and deliver bioactive ingredients. Alternatively, for applications where the lactoferrin itself is used as a functional component it may be advantageous to deliver the protein as nanoparticles rather than as individual molecules. One would expect that the protein nanoparticles would be less reactive than the individual protein molecules due to their lower surface area, yet still be in a form that could be conveniently incorporated into commercial products. Acknowledgments The authors gratefully acknowledge the financial support from the MICINN (José Castillejo Programme) of Spain. I.P. would also like to thank “Dirección General de Investigación del Ministerio de Ciencia y Tecnología” and “Universidad Politécnica de Valencia” (Spain) for the financial support of this investigation. This material is partly based upon work supported by United States Department of Agriculture, CREES, NRI Grants, and Massachusetts Department of Agricultural Resources. We also acknowledge funding from the University of Massachusetts (Hatch). References

Fig. 6. Influence of ionic strength (NaCl, 10e200 mM) in the thermal resistance (20 min, 75
C) of a 0.2% bLf solution, pH 7, adding the salt after (a) and before (b) heating.

Abe, H., Saito, H., Miyakawa, H., Tamura, Y., Shimamura, S., Nagao, E., et al. (1991). Heat stability of bovine lactoferrin at acidic pH. Journal of Dairy Science, 74, 65e71. Anderson, B. F., Baker, H. M., Norris, G. E., Rice, D. W., & Baker, E. N. (1989). Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 Å resolution. Journal of Molecular Biology, 209, 711e734. Benichou, A., Aserin, A., & Garti, N. (2002). Protein-polysaccharide interactions for stabilization of food emulsions. Journal of Dispersion Science and Technology, 23, 93e123. Brisson, G., Britten, M., & Pouliot, Y. (2007). Heat-induced aggregation of bovine lactoferrin at neutral pH: effect of iron saturation. International Dairy Journal, 17, 617e624. Brock, J. H. (1997). Lactoferrin structure function relationships: an overview. In T. W. Hutchens, & B. Lonnerdal (Eds.), Lactoferrin interactions and biological functions (3e25). Totowa, NJ, USA: Humana Press. Brock, J. H. (2002). The physiology of lactoferrin. Biochemistry and Cell Biology, 80(1), 1e6. Bromley, E. H. C., Krebs, M. R. H., & Donald, A. M. (2006). Mechanisms of structure formation in particulate gels of beta-lactoglobulin formed near the isoelectric point. European Physical Journal E, 21(2), 145e152. Chen, L., Remondetto, G. E., & Subirade, M. (2006). Food protein-based materials as nutraceutical delivery systems. Trends in Food Science & Technology, 17, 272e283.

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