Effect of high pressure homogenisation on microbial inactivation, protein structure and functionality of egg white

Food Research International 62 (2014) 718–725

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Effect of high pressure homogenisation on microbial inactivation, protein structure and functionality of egg white Agnese Panozzo a, Lara Manzocco b,⁎, Sonia Calligaris b, Ingrid Bartolomeoli b, Michela Maifreni b, Giovanna Lippe b, Maria Cristina Nicoli b a b

Laboratory of Food Technology, KU Leuven, Kasteelpark Arenberg 22, 3001 Heverlee, Belgium Dipartimento di Scienze degli Alimenti, Università di Udine, via Sondrio 2/A, 33100 Udine, Italy

a r t i c l e

i n f o

Article history: Received 6 March 2014 Accepted 19 April 2014 Available online 26 April 2014 Keywords: High pressure homogenisation Egg white Protein structure Foam Gel Immunoreactivity

a b s t r a c t The effect of high pressure homogenisation (HPH) on decontamination, protein structure (turbidity, particle size, free sulfhydryl groups, protein electrophoretic mobility) and selected functional properties (immunoreactivity, viscosity, foaming and gelling properties) of egg white were evaluated. HPH at 150 MPa for multiple passes allowed the progressive inactivation of Salmonella enterica SDMZ 9898. In addition, HPH modified egg white proteins by inducing unfolding and aggregation phenomena. The latter would occur by means of hydrophobic interactions among partially unfolded proteins. Protein structure modifications induced by HPH decreased egg white immunoreactivity probably due to protein epitopes hiding upon aggregation. HPH was not sufficient to modify egg white foaming properties. Depending on the intensity of the process, HPH allowed the modification of apparent viscosity of egg white and firmness of egg white gel. © 2014 Elsevier Ltd. All rights reserved.

Introduction In recent time, the demand for liquid egg products has been growing quickly due to their convenient format and easy use at both industrial and domestic levels. As known, pasteurisation is essential to obtain safe liquid egg products by destroying spoilage microorganisms and pathogens. Salmonella, mainly the egg-associated serotype Enteritidis, is the pathogen of concern. To address the issues of Salmonellosis caused by consumption of contaminated liquid eggs, European Regulation CEE 1441/2007 requires the absence of Salmonella in 25 g of product. The US Department of Agriculture has also established egg pasteurisation standards to produce Salmonella free egg products. In the case of liquid egg white, the product should be held for a minimum of 3.5 min at a process temperature of 56.7 °C (USDA 1969) or at 55.6 °C for at least 6.2 min (Code of Federal Regulations, 2010). Heat treatment is well known to impair the multifunctional properties of egg white, including its ability to coagulate, make foams upon whipping, beget emulsions, increase binding adhesion and contribute to the texture of various foods such as bakery products, meringues, meat products and cookies (Cunningham, 1995). Heating is also known to modify the nutritional and biological properties of egg white, including its digestibility and allergenicity. To this regard, protein

⁎ Corresponding author. Tel.: +39 0432 558152; fax: +39 0432 558130. E-mail address: [email protected] (L. Manzocco).

http://dx.doi.org/10.1016/j.foodres.2014.04.051 0963-9969/© 2014 Elsevier Ltd. All rights reserved.

cross-linking, deriving from the development of Maillard reaction, not only causes the loss of essential amino acids, but makes protein aggregates less accessible to digestive enzymes. In addition, heating may induce loss of conformational epitopes, giving reason to the changes in egg white immunoreactivity (Martos, Lopez-Exposito, Bencharitiwong, Berin, & Nowak-Wegrzyn, 2011). Several non-thermal technologies have been proposed for egg white pasteurisation, including high hydrostatic pressure, pulsed electric fields, UV-C light, pulsed light and microfiltration (Bridgman, 1914; Dunn, 1996; Jeantet, Baron, Nau, Roignant, & Brule, 1999; Mukhopadhyay, Tomasula, Luchansky, Porto-Fett, & Call, 2010; Unluturk, Atilgan, Baysal, & Tari, 2008). In this context, high pressure homogenisation (HPH) is a promising technique, particularly suitable for continuous production of fluid foods, allowing the limitation of thermal damage (Popper & Knorr, 1990). In general terms, during HPH, the fluid is forced through a narrow gap in the homogenizer valve, where it is submitted to a rapid acceleration (Dumay et al., 2013; Floury, Legrand, & Desrumaux, 2004). As a consequence, phenomena such as cavitation, shear and turbulence are simultaneously induced (Freudig, Tesch, & Schubert, 2003; Paquin, 1999), leading to an instant temperature increase whose magnitude depends on the intensity of the applied pressure. High pressure homogenisation has been demonstrated to cause inactivation of bacteria and yeasts in several different foods (Cruz et al., 2007; Diels & Michiels, 2006; Lanciotti, Gardini, Sinigaglia, & Guerzoni, 1996; Lanciotti, Sinigaglia, Angelini, & Guerzoni, 1994). However no indication is available about the possibility to exploit this technology to decontaminate egg white. Recently, Patrignani et al. (2013) demonstrated the possibility

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to efficiently inactivate Salmonella enterica in liquid whole egg by HPH at 100 MPa for up to 5 passes. These authors also observed minor impact on product viscosity as well as an increase in foam capacity and stability. At similar pressure levels, the typical egg lipoprotein matrix was maintained and no proteolysis was detected in high pressure homogenised whole egg (Marco-Molés, Hernando, Llorca, & Pérez-Munuera, 2009). Despite these evidences, the effect of high pressure homogenisation on protein structure and functions is still contradictory. For instance, Speroni et al. (2005) reported HPH at 200–600 MPa to induce denaturation and subsequent aggregation of egg lipoproteins. On the other hand, Subirade, Loupil, Allain, and Paquin (1998) observed that HPH treatment at 140 MPa did not produce changes in the secondary structure of β-lactoglobulin but promoted slight interactions among particles. In the case of whey proteins, dissociation of large aggregates was observed resulting in an increase of surface hydrophobicity (Bouaouina, Desrumaux, Loisel, & Legrand, 2006). Upon homogenisation at higher pressures (N 250 MPa), whey protein aggregation was also observed (Grácia-Juliá et al., 2008). Similarly, soybean 11S globulins were reported to denature upon high pressure homogenisation leading to the formation of a gel-like architecture (Floury, Desrumaux, & Legrand, 2002). In the light of these considerations, it can be inferred that high pressure homogenisation could modify the structure of egg white proteins, potentially affecting their functional properties. The effect of HPH on protein structure could be emphasised by either increasing homogenisation pressure or applying multiple passes at moderate pressure. In this paper, we investigated the effect of high pressure homogenisation on S. enterica inoculated in egg white and on protein structure and functionality. To this purpose, egg white was submitted to multi-pass homogenisation at 150 MPa. Samples were analysed for microbial inactivation as well as for protein structural changes by evaluating turbidity, light scattering properties, free sulfhydryl content and electrophoretic pattern. The effect of protein structural changes on some technological properties (viscosity, foam ability and stability, and gel firmness) as well as on immunoreactivity of egg white were then evaluated.

Materials and methods Sample preparation Organic eggs were obtained from a local supermarket. The egg shell was cleaned with a hydroalcoholic solution (ethanol 70% v/v) and wet shells were allowed to air dry for a few minutes before aseptic breaking. The egg white was manually separated from the yolk and the chalazae were removed. About 10–12 eggs were used to yield approximately 250 mL of egg white. The albumen was gently stirred in a sterilised beaker. Egg white pH was 8.6 ± 0.3 (pH-metre Crison MicropH 2001, Modena, Italy; glass electrode Crison pH 2–11, Alella, Spain).

719

Temperature The sample temperature was measured just before and immediately after homogenisation by a copper-constantan thermocouple probe (Ellab, Denmark) connected to a portable data logger (mod. 502A1, Tersid, Milano, Italy). Bacterial strain and inoculation S. enterica subsp. enterica 9898 DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) was used in this study. The strain added with 50% (v/v) sterile glycerol as cryoprotectant was stored at −80 °C in Triptone Soya Broth (TSB) (Oxoid, Milano, Italy) until its use. Strains were incubated in TSB at 37 °C per 24 h and subsequently plated onto Tryptone Soya Agar (TSA, Oxoid, Milano, Italy). A single colony was inoculated into TSB and incubated at 37 °C for 24 h. Subsequently, the culture was centrifuged at 13,000 rpm for 10 min and the pellets were washed. Inoculation was made by resuspending the pellets in Maximum Recovery Diluent (MRD, Oxoid, Milano, Italy). An aliquot of this suspension was added to egg white to obtain a final concentration about 106 CFU mL−1. Inoculated egg white was submitted to high pressure homogenisation (HPH) as previously described, and immediately analysed for viable counts of Salmonella. Microbiological analyses Xylose Lysine Deoxycholate (XLD, Oxoid, Milano, Italy) and Plate Count Agar (PCA, Oxoid) were used to assess Salmonella by pour plating, after making appropriate serial dilutions with peptone water. Salmonella was counted after 24 h at 37 °C and 36–48 h at 30 °C on XLD and PCA, respectively. PCA medium was also used to evaluate possible contamination of samples and the injured cells. Preliminary trials were also carried out on untreated egg white to evaluate the presence/absence of naturally occurring Salmonella. In particular, 25 mL of egg white was diluted with 225 mL of buffered peptone water (BPW, Oxoid, Milano, Italy), homogenised in Stomacher (PBI, Milano, Italy) for 2 min and incubated at 37 °C for 24 h. 0.1 mL of BPW was added with 9.9 mL Rappaport Vassiliadis (RV, Oxoid, Milano, Italy) and incubated at 42–43 °C for 18–24 h. The presence/absence of S. enterica was checked by spreading onto XLD plates and incubated at 37 °C for 24 h. Each experiment was replicated 3 times. Data are reported as mean values and standard deviations. Optical density Optical density at 680 nm was measured at 25 °C by a UV–vis spectrophotometer (UV-2501 PC, Shimadzu Kyoto, Japan) with a 1 cm path-length cuvette. Particle size

High pressure homogenisation Egg white was homogenised using a two stage high pressure homogeniser (Panda PLUS 2000, Gea Niro Soavi, Parma, Italy) provided with cylindrical tungsten carbide homogenising valves. The first valve, which is the actual homogenisation stage, was set at 20, 50, 100, and 150 MPa and the second one at 5 MPa. Aliquots of 250 mL egg white were homogenised at 20, 50, 100, 150 MPa via single pass and at 150 MPa via multiple passes up to 17 at 10.8 L/h flow rate. The homogeniser inlet and outlet were connected to a heat exchanger (Julabo F70, Seelbach, Germany) set at 4 °C. Additional samples were obtained by homogenising the egg white with the first valve set at 0 MPa via 1–17 passes. Untreated egg white was considered as control. All the samples were kept at 4 °C until analysis. All the samples were analysed within 24 h after the homogenisation process.

Light scattering measures were made using a Particle Sizer NICOMPTM 380 ZLS (PSS NICOMP Particle Sizing System, Santa Barbara, California, USA). Samples were diluted 1:50 (v/v) with deionised water. The angle of observation was 90°. The refractive index of the solution was set at 1.333 and the viscosity was approximated to that of pure water at 25 °C. Hydrodynamic diameter refers to the corresponding volume distribution calculated by NICOMP Distribution Analysis. Free sulfhydryl content The concentration of free sulfhydryl groups (SH) of the egg white samples was determined using Ellman's reagent (5′,5-dithiobis (2-nitrobenzoic acid), DTNB) (Sigma Aldrich. Milan, Italy). Changes in free sulfhydryl groups were measured in triplicate as reported by Beveridge, Toma, and Nakai (1974). Briefly, egg white (1.5 g) was diluted

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to 10 mL with 1% (w/v) NaCl in Tris-glycine buffer (1.04% Tris, 0.69% glycine, 0.12% EDTA (w/v), pH 8.0) (Sigma Aldrich, Milan, Italy). After 15 min, absorbance was measured at 412 nm by a UV–vis spectrophotometer (UV-2501 PC, Shimadzu Kyoto, Japan). Concentration of free sulfhydryl groups (μM g−1) was calculated from the following equation: 73:53  A412  D SH ¼ C

ð1Þ

where A412 is the absorbance at 412 nm; C is egg white concentration 6 10 (mg mL−1); D is the dilution factor; and 73.53 is derived from 1:3610 −4 ; 4 1.36 · 10 is the molar absorptivity (Ellman, 1959). SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (1970) on a Mini 2-D (Bio-Rad, Richmond, California, USA) vertical electrophoresis cell. Egg white was diluted 1:4 with 0.05 M Tris–HCl buffer pH 8.8 containing 2.34% (w/v) NaCl. The egg white solution was then further diluted 1:4 with sample buffer (Bromophenol blue 0.01%, 120 mM Tris–HCl, pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol). Samples of 5 μL containing 15 μg of proteins were loaded on a 15% (w/v) polyacrylamide gel. The egg white electrophoretic patterns were obtained by running the samples at 15 mA for 1.5 h. Standard proteins of known molecular weight were used (Precision Plus Protein™ Dual Color Standards, Bio-Rad, Segrate, Italy). The gels were subsequently stained in NOVEX® Colloidal Blue Staining Kit (Invitrogen, Carlsbad, California, USA) for one night and then destained in bi-distilled water. Gels were scanned and analysed with the ImageQuant TL Image Analysis Software (Amersham Bioscience Inc., Piscataway, New Jersey, USA) to determine the molecular weight of each band. Immunoreactive egg white proteins The quantitative determination of immunoreactive egg white was performed at 25 °C by the enzyme immunoassay RIDASCREEN® Fast Ei/Egg Protein (R-Biopharm AG, Darmstadt, Germany) according to the manufacturer's recommendations. Analyses were performed on samples diluted 1:108 with the 0.05 M Tris–HCl pH 8.8 buffer containing 2.34% (w/v) NaCl. Absorbance at 450 nm was measured by using a microplate reader (Sunrise, Tecan Group, Männedorf, Switzerland). Concentration of immunoreactive egg white was determined according to the calibration curve obtained using the standard solutions provided with the immunoassay kit. Apparent viscosity Viscosity at 20 °C was measured by a Stresstech Rheometer (Reologia Instruments AB, Lund, Sweden) with a parallel plate geometry of 40 mm diameter, and a gap of 2 mm. The system was thermostated by a Jumo Dicon cell (Sm M.K. Juccheim GMBM & Co, Fulda, Germany). The measurements were performed at shear rate from 1.093 to 159.4 s−1. Apparent viscosity was calculated at shear rate equal to 5.17 s−1. Foam ability and stability Foams were obtained by whipping 5 mL of egg white for 3 min at 20 °C in a 50 mL cylinder by a high speed mixer (Ika-Werke, DI 25 Basic, Staufen, Germany) operating at 9500 rpm. The volume of the foam and of the drained liquid was assessed just after whipping and during holding up to 30 min at 20 °C. Percentage foam ability (FA) and stability (FS) were calculated as follows: FA ð%Þ ¼ ðVf−V0 Þ=V0  100

ð2Þ

FS ð%Þ ¼ Vf 30 =Vf  100

ð3Þ

where Vf is the foam volume, V0 is the initial volume of the liquid egg white and Vf30 is the foam volume after 30 min observation. Microscopy Just whipped foams and foams held for 30 min at 20 °C were placed onto a microscope slide. Pictures of foam bubbles were acquired as quickly as possible using a digital camera (Leica EC3, Solms, Germany) mounted on a Leica™ microscope (Leica DM 2000, Solms, Germany), magnification 200×. Gel firmness Aliquots of 3.5 g of egg white were introduced in 10 mL capacity vials, hermetically sealed with butyl septa and metallic caps. Samples were heated in a water bath (Yellow Line, IKA-Werke, Germany) at 90 °C for 5 min. Firmness was measured by a puncture test using an Instron 4301 (Instron LTD, High Wycombe, UK). The instrumental settings and operations were accomplished using the software Automated Materials Testing System (version 5, Series IX, Instron LTD, High Wycombe, UK). Samples were punctured with a 1.5 mm cylindrical probe at a crosshead speed of 10 cm min−1. Force–distance curves were obtained from the puncture tests and firmness was taken as the maximum load (N) required to puncture the gel 0.5 cm. For each sample 6 measures were performed. Statistical analysis The results are averages of at least three measurements taken from different samples and are reported as means ± SD. Analyses of variance (ANOVA) was performed with a significance level set to p b 0.05 (Statistica for Windows, ver. 5.1, Statsoft Inc. Tulsa, USA, 1997). The Tukey procedure was used to test for differences between means. Results and discussion The effect of HPH on S. enterica inoculated in egg white and on egg white protein structure and functionality was studied. Preliminary trials were performed by homogenising egg white at 20, 50, 100, and 150 MPa for a single pass. However, these processing conditions were not sufficient to observe changes in egg white protein structure (data not shown). For this reason, egg white was subjected to HPH at 150 MPa for multiple passes. In particular, egg white was cooled at 4 °C and homogenised at 150 MPa for up to 17 passes. Untreated egg white and egg white homogenised at 0 MPa for up to 17 passes were taken as controls. In order to minimise the increase in temperature during processing, the inlet and outlet of the homogeniser were connected to a cooling system set at 4 °C. Operating under these conditions, no temperature changes in egg white were detected upon homogenisation at 0 MPa for multiple passes. By contrast, homogenisation at 150 MPa promoted a progressive increase in egg white temperature (Fig. 1). The latter never exceeded 35 °C, even in the case of the highest number of passes (Hatta, Kitabatake, & Doi, 1986). Being this temperature far lower than the denaturation temperature of ovotransferrin (61 °C), which is the most heat-sensitive egg white protein (Mine, 1995), any changes that have occurred in the samples can be reasonably attributed to the sole effect of the HPH process. Inactivation of S. enterica 9898 DSMZ The microbial inactivation effect of high pressure homogenisation was evaluated by processing egg white previously inoculated with about 6 log CFU mL−1 of S. enterica 9898 DSMZ. The initial bacterial concentration used in this study was much higher than the one that would ever be encountered in commercial products. Nevertheless, it was chosen to better understand the inactivation effect. The results of the

A. Panozzo et al. / Food Research International 62 (2014) 718–725

35 30

0

2

4

8

12

17

0.5

ab

ab

a

25 0.4

20

b

15

c

O.D. 680 nm

Temperature (˚C)

Control

a

a ab

721

10 5

d

0 Control

0 MPa

2 passes

4 passes

0.3

c 0.1

d 0 Control

microbiological analyses are reported in Table 1. Homogenisation at 150 MPa for multiple passes resulted in the progressive inactivation of inoculated S. enterica 9898 DSMZ. After 4 passes, more than 3.5 log-cycles of inactivation were achieved. The increase in the number of passes up to 8 significantly affected microbial inactivation so that reductions of about 4.7 and 5 log-cycles were noted on CBT agar and XLD, respectively. A further increase in passes up to 17 produced a reduction of 5 log-cycles on PCA, while counts lower than the detection limit (10 CFU mL−1) were observed on XLD. For each homogenisation treatment, counts on the selective XLD medium tended to be lower than those detected on the non-selective PCA one. It is likely that HPH induced not only Salmonella inactivation but also sublethal injury following mechanical stress. Injured Salmonella cells are probably allowed the recovery by the use of a non-selective medium, avoiding overestimation of the HPH antimicrobial effect. Similar inactivation levels were observed by Patrignani et al. (2013) when liquid whole egg, previously inoculated with 7 log S. enterica serovar Enteritidis 155, was homogenised at 100 MPa for 5 passes. This difference might depend not only on the type of microorganism, but also on several other factors, including HPH valve geometry as well as sample temperature and rheological properties. In addition, HPH at 150, 200, and 250 MPa were found to progressively decrease the viable count of S. enterica serovar Senftenberg 775W inoculated in whole liquid egg (Velazquez-Estrada, Hernandez-Herrero, Lopez-Pedemonte, GuamisLopez, & Roig-Sagues, 2008). According to these authors, HPH at 250 MPa induced total and 3.2 log CFU mL−1 inactivation of S. enterica in liquid whole egg inoculated with approximately 3 and 7 log CFU mL−1 respectively. Egg white protein structure High pressure homogenisation induced changes in egg white optical density at 680 nm (Fig. 2). An initial decrease in optical density after

Table 1 Counts of S. enterica 9898 DSMZ on Plate Count agar (PCA) and XLD agar (XLD), inoculated in egg white (control) and homogenised at 150 MPa via multiple passes (2, 4, 8, 12, 17 passes). S. enterica (log CFU mL−1) PCA Control 2 passes 4 passes 8 passes 12 passes 17 passes

6.48 3.90 2.86 1.76 1.69 1.46

XLD ± ± ± ± ± ±

0.20a 0.02b 0.07c 0.15d 0.21d 0.28d

6.38 ± 3.74 ± 2.66 ± 1.36 ± 1.20 ± bd.l.

0.04a 0.12b 0.19c 0.10d 0.17d

d.l.: detection limit (10 CFU mL−1). a,b,c,d Means on the same column indicated by the same letter are not statistically different (P ≥ 0.05).

c

0.2

8 passes 12 passes 17 passes

Fig. 1. Egg white temperature of untreated egg white (control), egg white homogenised at 0 MPa (0 MPa), and egg white homogenised at 150 MPa via multiple passes (2, 4, 8, 12, 17 passes). a,b,c,dBars indicated by the same letter are not statistically different (P ≥ 0.05).

Sample

b,c

c

0 MPa

2 passes

4 passes

8 passes 12 passes 17 passes

Fig. 2. Optical density (O.D.) at 680 nm, and visual appearance (inset) of untreated egg white (control), egg white homogenised at 0 MPa (0 MPa), and egg white homogenised at 150 MPa via multiple passes (2, 4, 8, 12, 17 passes). Bars indicated by the same letter are not statistically different (P ≥ 0.05). (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)

homogenisation at 0 MPa was observed. Optical density of egg white homogenised at 0 MPa refers to the sample homogenised for one single pass since no effect of the number of passes (up to 17) at 0 MPa was detected. On the other hand, a progressive increase in optical density upon multiple homogenisation passes at 150 MPa was noted. These changes were accompanied by a progressive shift of egg white colour from yellow towards milky white (Fig. 2, inset), suggesting the formation of protein aggregates/network able to scatter light. Dynamic light scattering analysis (Table 2) of untreated egg white showed the presence of two classes of particles with an average diameter of about 143 nm and larger than 8000 nm. The latter was the main class of particles in the sample and accounted for protein aggregates formed through non-covalent weak interactions. It is well known that in fresh egg white ovomucin, which is a highly glycosylated protein with a very high molecular weight, can aggregate with other egg white proteins by means of electrostatic interactions. In particular, the carboxylic groups of the ovomucin sialic acids can interact with the amino group of lysozyme lysine residues to form a lysozyme–ovomucin complex that would be responsible for the gel-like structure of egg white (Kato, Imoto, & Yagashita, 1975; Mine, 1995). These weak interactions would be easily broken by a single homogenisation pass at 0 MPa, leading to a significant increase in the presence of particles with an average diameter equal to 143 nm as well as to the formation of smaller particles with an average diameter of about 35 nm. No significant differences in the size distribution of protein particles were observed upon further homogenisation at 150 MPa up to 8 passes. By contrast, egg white homogenised at 150 MPa for 12 and 17 passes showed the occurrence of a novel class of particles with an average diameter equal to 473 nm. The latter might account for HPH-induced protein unfolding/aggregation. To investigate the nature of HPH-induced protein structural modifications, samples were analysed for the concentration of free sulfhydryl groups (SH) (Fig. 3). In fact, based on literature data, the decrease in free SH could indicate protein cross-linking, while their increase generally accounts for both protein unfolding and backbone fragmentation (Beveridge et al., 1974). In the case of egg white homogenised at 0 MPa via a single pass, an increase in free SH was observed. Such increase can be consistent with the exposure of free SH upon disruption of the large protein aggregates having hydrodynamic radius higher than 8000 nm (Table 2). With regard to the egg white homogenised at 150 MPa, after 2 homogenisation passes an intermediate content in free SH between the control and the egg white homogenised at 0 MPa was observed. On the other hand, further homogenisation passes led to a concentration of

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A. Panozzo et al. / Food Research International 62 (2014) 718–725

Table 2 Percentage distribution of particles with average hydrodynamic diameter (ø) equal to 13, 35, 143, 473, and larger than 8000 nm in untreated egg white (Control), egg white homogenised at 0 MPa (0 MPa), and egg white homogenised at 150 MPa via multiple passes (2, 4, 8, 12, 17 passes). Particles (%)

Control 0 MPa 2 passes 4 passes 8 passes 12 passes 17 passes

ø ≈ 13 nm

ø ≈ 35 nm

ø ≈ 143 nm

ø ≈ 473 nm

ø N 8000 nm

n.d. n.d. n.d. 3.47 ± 1.25 n.d. 1.40 ± 0.66 0.80 ± 0.01

n.d. 5.37 ± 1.79 4.75 ± 1.64 n.d. 3.65 ± 0.64 n.d. n.d.

14.70 84.90 92.02 90.82 85.30 40.90 49.67

n.d. n.d. n.d. n.d. n.d. 58.43 ± 4.65 56.85 ± 6.01

87.44 ± 9.59 n.d. n.d. n.d. n.d. n.d. n.d.

± ± ± ± ± ± ±

2.19 13.19 4.34 6.19 4.63 4.16 10.07

n.d.: not detected.

free SH similar to that of the egg white homogenised at 0 MPa. The initial decrease and subsequent increase in free SH upon multiple homogenisation passes at 150 MPa supports the hypothesis of protein structural modification by means of both unfolding and aggregation phenomena. The latter would be concomitantly induced by HPH, leading to counterbalancing effects on the changes in free SH. Two different mechanisms have been suggested for protein aggregation. First, the exposure of the hydrophobic regions as a consequence of protein unfolding can lead to hydrophobic interactions (Rao & Labuza, 2012). Second, sulfhydryl groups, which are buried in the native state of the protein, can become available in the unfolded proteins and react to form intermolecular cross-links (Ferry, 1948). In the present case, the reversible changes in free SH groups are more likely attributable to the formation and breakage of hydrophobic interactions, rather than to protein crosslinking. In fact, weak interactions such as the hydrophobic ones can be easily formed and broken upon HPH. Instead, covalent bonds responsible for protein crosslinking are known to be hardly affected by HPH (Subirade et al., 1998). To confirm this hypothesis egg white samples were analysed by SDS-PAGE (Fig. 4). As known, SDS is an anionic detergent able to disrupt non-covalent bonds, such as hydrophobic interactions among proteins, and impart a negative charge to proteins, which are separated according to their molecular weight. Moreover, no reductant was added to the samples, to reveal eventual protein aggregation due to S–S formation. The SDS-PAGE pattern of the untreated egg white exhibited three main bands that, by comparison with the standard molecular weights (MW) and literature information (Awadé & Efstathiou, 1999; Guerin-Dubiard et al., 2006; Mine, 1995), were attributed to ovotransferrin, ovalbumin/ovomucoid, and lysozyme. Whatever the pressure level, no difference between the SDS-PAGE patterns of the homogenised egg white samples and that of the untreated egg white was observed. Therefore, polyacrylamide gel electrophoresis under dissociating conditions confirmed that HPH-induced aggregation occurred mainly through new, possibly hydrophobic interactions and

Fig. 3. Free SH groups in untreated egg white (control), egg white homogenised at 0 MPa (0 MPa), and egg white homogenised at 150 MPa via multiple passes (2, 4, 8, 12, 17 passes). Bars indicated by the same letter are not statistically different (P ≥ 0.05).

not by the formation of covalent bonds. Similar results were also reported by Grácia-Juliá et al. (2008) for whey protein dispersions homogenised at pressures higher than 250 MPa, and by Marco-Molés et al. (2009) for liquid whole egg homogenised at 100 MPa for 3 cycles. Results relevant to the distribution of the particle size (Table 2), free SH (Fig. 2) and protein fractions (Fig. 4) suggest HPH to promote structural modifications of egg white proteins by means of unfolding and aggregation phenomena. In particular, homogenisation initially induced partial unfolding of egg white protein leading to exposure of protein SH-groups. On further homogenisation partial aggregation of unfolded proteins also took place, much probably through hydrophobic interactions. Egg white protein functions To understand whether the HPH-induced unfolding/aggregation among egg white proteins could be associated to modifications in their functionality, samples were analysed for their immunoreactivity. In fact, despite the great nutritional relevance of egg white, it also represents a critical issue for its allergenic potential. Ovalbumin, ovomucoid, lysozyme, and ovotranferrin are the major allergenic proteins of egg white (Mine & Yang, 2008). However, literature evidences report that thermal treatments and non-thermal processes can modify egg white allergenicity (Manzocco, Panozzo, & Nicoli, 2012, 2013; Mine & Yang, 2008). Based on these considerations, egg white samples processed by HPH were analysed for immunoreactivity by an ELISA method (Fig. 5). A slight, but significant decrease in immunoreactivity was observed upon HPH processing of egg white. Protein immunoreactivity was reduced by circa 10% when egg white was homogenised at 150 MPa for 4 or more passes. This suggests that HPH induced protein unfolding and aggregation might partially hide protein epitopes, leading to the decrease of immunoreactivity. Samples were also analysed for apparent viscosity, gelling and foaming properties (Table 3). Apparent viscosity, taken as an indicator of the rheological behaviour of egg white, was found to decrease after a single homogenisation pass at 0 MPa. This effect can be attributed to the disruption of the weak and unstable network characterising the large protein aggregates naturally occurring in the untreated egg white (Table 2). Upon further homogenisation passes at 150 MPa, viscosity values progressively approached that of the untreated egg white (Table 3), reasonably due to the formation of a novel protein network by weak hydrophobic interactions. Gel firmness, defined as the maximum load needed to puncture the gel, was also analysed as an indicator of the textural properties of egg white gels (Table 3). The firmest gel was obtained from the egg white homogenised at 0 MPa. By contrast, the least firm gel was obtained from the egg white homogenised at 150 MPa for 2 passes. Although on further homogenisation passes a progressive increase in gel firmness was observed, the egg white homogenised at 150 MPa always produced softer gels than those obtained from the untreated and 0 MPa

A. Panozzo et al. / Food Research International 62 (2014) 718–725

MW

Control

0 MPa

723

2 passes 4 passes 8 passes 12 passes 17 passes

250 kDa 150 kDa 100 kDa 75 kDa

Ovotransferrin

50 kDa

Ovalbumin/ Ovomucoid

37 kDa 25 kDa 20 kDa

Lysozyme

15 kDa

10 kDa

Fig. 4. Electrophoretic pattern of untreated egg white (control), egg white homogenised at 0 MPa (0 MPa), and egg white homogenised at 150 MPa via multiple passes (2, 4, 8, 12, 17 passes). Molecular weight standards (MW) are in the far left lane.

homogenised egg white. Similarly, a decrease in gel stiffness (expressed as G′) was also observed upon HPH of soybean proteins (Speroni, Jung, & de Lamballerie, 2010). As known, the thermal transformation of egg white proteins into a gel involves partial protein unfolding with exposure of functional groups. Thereafter, ovalbumin, ovotransferrin and lysozyme form intermolecular β-sheet structures through hydrogen-bonding, electrostatic and hydrophobic interactions, resulting in a gel network (Mine, 1995). The strength of the protein gel network was reported to be dependent on the balance between protein hydrophobic interactions and electrostatic repulsive forces. In particular, the better the balance between electrostatic and hydrophobic interactions, the stronger the gel (Hatta et al., 1986). The decrease in particle dimensions (Table 2) in combination with the evidence of protein unfolding (Fig. 3) after homogenisation at 0 MPa might induce a better balance between electrostatic and hydrophobic interactions leading to a firmer gel. Upon homogenisation at 150 MPa both protein unfolding and aggregation phenomena could unbalance the attractive and repulsive forces between egg white proteins, favouring the formation of softer gels. With regard to foaming properties (Table 3), no differences were observed in foam ability of HPH-treated egg white. Foams obtained from untreated and homogenised egg white samples were also held during 30 min at room temperature to evaluate foam stability. As expected, foam stability of all the samples was found to decrease after 30 min

resting. The foam stability of the untreated egg white after 30 min was about 86% of the just prepared foam and no changes in egg white foam stability were observed upon homogenisation at both 0 and 150 MPa. These results were supported by microscopy analysis of just prepared and 30 min held egg white foams. As an example micrographs relevant to the foams obtained from the untreated egg white, egg white homogenised at 0 MPa, and egg white homogenised at 150 MPa for 17 passes are presented in Fig. 6. Microscopy images clearly show that no changes in bubble size could be observed among the samples for both just prepared foams and 30 min held foams.

Conclusions The present results demonstrate that HPH can be regarded as a promising alternative to thermal pasteurisation of egg white. Contrarily to thermal treatments, HPH could be exploited to obtain safe egg white without impairing its multifunctional properties. In fact, under the actual experimental conditions, multiple HPH passes induced protein structural modifications by promoting partial aggregation, possibly due to protein unfolding and exposure of hydrophobic regions. These structural changes resulted in the formation of a weak and unstable protein network, that did not affect egg white foaming properties. In addition, by choosing proper processing conditions, some egg white functionalities, such as immunoreactivity and gel firmness, can be steered to obtain egg white with specific functional properties.

Table 3 Apparent viscosity, gel firmness, foam ability and stability of untreated egg white (control), egg white homogenised at 0 MPa (0 MPa), and egg white homogenised at 150 MPa via multiple passes (2, 4, 8, 12, 17 passes). Apparent viscosity (Pa s)

Fig. 5. Immunoreactive proteins in untreated egg white (control), egg white homogenised at 0 MPa (0 MPa), and egg white homogenised at 150 MPa via multiple passes (2, 4, 8, 12, 17 passes). Bars indicated by the same letter are not statistically different (P ≥ 0.05).

Control 0 MPa 2 passes 4 passes 8 passes 12 passes 17 passes a,b,c,d,e

0.057 0.035 0.047 0.050 0.055 0.057 0.055

± ± ± ± ± ± ±

0.005 a 0.006 b 0.006 a,b 0.001 a,b 0.007 a 0.005 a 0.013 a

Gel firmness (N) 0.148 0.181 0.067 0.080 0.095 0.110 0.118

± ± ± ± ± ± ±

0.016 b 0.022 a 0.003 e 0.005 e 0.007 d,e 0.002 c,d 0.002 c

Foam ability (mL) 18.7 19.0 19.0 18.7 17.7 18.7 18.7

± ± ± ± ± ± ±

0.6 a 1.0 a 1.0 a 0.6 a 0.6 a 0.6 a 0.6 a

Foam stability (%) 86.1 89.1 84.0 86.0 88.7 85.2 84.3

± ± ± ± ± ± ±

4.1 a 7.1 a 7.1 a 3.8 a 4.4 a 3.3 a 6.3 a

Means on the same column indicated by the same letter are not statistically different (P ≥ 0.05).

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Control (A)

Control (B)

0 MPa (A)

0 MPa (B)

17 passes (A)

17 passes (B)

Fig. 6. Micrographs (200×) of just prepared (A) and 30 min held (B) foams obtained from untreated egg white (control), egg white homogenised at 0 MPa (0 MPa), and egg white homogenised at 150 MPa via 17 passes.

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