Effect of pulsed electric field and thermal treatment on the physicochemical properties of lactoferrin with different iron saturation levels

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Abstract

Bovine lactoferrin (LF) with different iron saturation levels (i.e., native (N-) LF, Apo-LF and Holo-LF) dissolved in simulated milk ultrafiltrate was treated by pulsed electric fields. Various heat treatments were conducted as comparisons. The concentration of LF, electrophoretic mobility of the proteins, surface hydrophobicity and the release of LF-bound ferric ions to the aqueous phase of the LF preparations after the PEF or thermal treatments were determined. PEF treatments did not affect the physicochemical properties of Apo-LF, N-LF, or Holo-LF at treatment temperatures ≤50, ≤60, ≤65 °C, respectively. Changes in LF properties at higher temperatures were largely or entirely due to concurrent thermal effects during PEF treatments. The thermal stability of LF correlated with the level of iron saturation. These results will be useful when developing a PEF process for microbial inactivation of LF-containing dairy products with the aim of maximising the retention of this heat-sensitive protein.

Introduction

Many studies have demonstrated that lactoferrin (LF) is a protein with biological activity, including iron-binding ability, antimicrobial, antiviral, anti-infective, immunomodulatory, and anti-inflammatory effects (Baker and Baker, 2009, Jenssen and Hancock, 2009, Ochoa and Cleary, 2009, Pan et al., 2006, Puddu et al., 2009). Fortification with LF has been applied in various foods, such as infant formulae, yoghurt and beverages, for its biological benefits (Tomita et al., 2009).

Traditional thermal processing to guarantee the food safety can induce undesirable denaturation of LF, thus impairing its biological activity (Brisson et al., 2007, Indyk et al., 2007, Kussendrager, 1994, Paulsson et al., 1993). Pulsed electric field (PEF) treatment has been developed as a non-thermal processing technology for the inactivation of microorganisms in liquid foods at low to moderately elevated temperatures (Mosqueda-Melgar et al., 2008, Toepfl et al., 2006).

One of the advantages of PEF over conventional thermal treatment is believed to be its effectiveness in microbial inactivation combined with few effects on heat-sensitive compounds. For example, Barsotti, Dumay, Mu, Fernandez-Diaz, and Cheftel (2001) found that PEF treatment at 21–33 kV cm−1 with 200 exponential decay pulses of a 1.2 μs pulse width (1.7–2.7 kJ pulse−1 mL−1, temperature not reported) did not cause detectable unfolding or aggregation of β-lactoglobulin (β-Lg, 2–16.7%, w/v, in phosphate buffer). Li, Bomser, and Zhang (2005) and Li, Zhang, Lee, and Pham (2003) reported no change in the content and secondary structure of bovine immunoglobulin G (IgG) in either soymilk enriched with hyperimmunised dairy milk protein concentrate or borate buffer after a PEF treatment at 41 kV cm−1 for 54 μs (specific pulsing energy input not reported, final temperature 43.8 °C), the condition under which microbial inactivation equivalent to that of the thermal pasteurisation was achieved.

Similar results were found with lactoperoxidase (LP), the key component of the natural antimicrobial lactoperoxidase-thiocyanate-hydrogen peroxide (LP-SCN-H2O2) system in raw milk. For instance, Grahl and Markl (1996) reported that PEF treatment of raw milk at 21.5 kV cm−1 with a specific energy input of 400 kJ L−1 and a final temperature of <50 °C caused less than 15% inactivation of LP. Similarly, Van Loey, Verachtert, and Hendrickx (2001) reported that there was no decrease in the activity of LP in raw milk treated by PEF at 19 kV cm−1 using up to 100 exponential decay pulses of a 5 μs pulse width with a total pulsing energy of up to 500 kJ kg−1 (temperature not reported). More recently, Jaeger and Knorr (2007) observed a <10% inactivation of LP in raw milk after PEF treatment at 35 kV cm−1 and 30 °C with a specific pulsing energy input of up to 220 kJ kg−1. These reports, although using varied conditions and PEF units, suggest that PEF treatment has negligible effects on the heat-sensitive bioactive dairy proteins. However, few studies have reported the effect of PEF treatment on LF. De Luis et al. (2009) reported that LF concentration in either raw milk or whey was not changed after PEF treatment at 37.6 kV cm−1 using 200 exponential pulses (treatment temperature <35 °C).

The use of PEF treatment in combination with sub-pasteurisation temperatures has the potential to achieve similar microbial inactivation but with shorter exposure time to high temperatures than that of the conventional thermal pasteurisation processing (Shamsi et al., 2008, Walkling-Ribeiro et al., 2009). However, the effect of PEF treatment at elevated temperatures on the integrity of LF has not been reported. The objectives of the present study were to investigate the effect of PEF treatment temperatures on the physicochemical properties of lactoferrin with different iron saturation levels at a range of low to sub-pasteurisation temperatures; to compare the effect of PEF with that of the thermal treatment, and, therefore, to provide critical information for considering PEF as a processing technology for microbial inactivation in dairy products.

Section snippets

Lactoferrin preparations

Three purified bovine LF samples with different iron saturation levels were obtained from Dr. J.-P. Perraudin of Biopole S.A. (Les Isnes, Gembloux, Belgium). The unmodified native (N-LF), iron-depleted (Apo-LF), and iron-saturated LF (Holo-LF) samples had protein contents of 98.3, 95.7, and 89.5%, and iron saturation levels of 24.5, 6, and 78.7%, respectively. Simulated milk ultrafiltrate (SMUF) was prepared using the formulation reported by Jenness and Koops (1962). The LF solutions (0.4 mg mL

Effect of PEF and thermal treatments on lactoferrin content

For the N-LF samples, both the LFA (0.38–0.39 mg mL−1) and LFE (0.36–0.37 mg mL−1) values remained constant for PEF (mono- and bi-polar) and non-PEF-control treated samples processed between 30 and 60 °C (Fig. 1a). However, increasing the treatment temperature to 65–70 °C decreased (P < 0.05) the LFA content to 0.34–0.37 mg mL−1 and LFE to 0.26–0.30 mg mL−1 for both of the PEF and the non-PEF treatments. All thermal treatments decreased (P < 0.05) the content of both LFA and LFE, with LFA

Discussion

Although a number of studies have reported the effect of thermal treatment on LF (Brisson et al., 2007, Indyk et al., 2007, Kussendrager, 1994, Paulsson et al., 1993, Rüegg et al., 1977), the effect of PEF treatment, especially combined with elevated temperatures, on this heat-sensitive bioactive milk protein has not been extensively studied. The current study investigated the effect of PEF in combination with low to elevated but sub-pasteurisation temperatures on the physicochemical properties

Conclusions

PEF treatment did not change any properties of Apo-LF, N-LF or Holo-LF at treatment temperatures of ≤50, ≤60, ≤65 °C, respectively. At higher temperatures, the LF concentration decreased, particularly for Apo-LF, but very little for Holo-LF. Some protein aggregation was observed by non-reducing SDS-PAGE at these elevated temperatures. The increase in surface hydrophobicity was pronounced, particularly for Holo-LF, but from the lowest base. A small amount of iron was released from Holo-LF

Acknowledgements

Qian (Sherry) Sui is a PhD candidate at the University of Melbourne with financial support from a Melbourne International Research Scholarship and a Victorian State Government Science and Technology Infrastructure Grant (STI-3) scholarship through the Innovative Foods Centre. We would like to thank Dr J.-P. Perraudin of Biopole S.A. for supplying the lactoferrin samples.

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Current address: National Center for Food Safety and Technology, Illinois Institute of Technology, Summit-Argo, IL 60501-1957, USA.

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