- Email: [email protected]
Effect of heating whey proteins in the presence of milk fat globule membrane extract or phospholipids from buttermilk
Accepted Manuscript Effect of heating whey proteins in the presence of milk fat globule membrane extract or phospholipids from buttermilk Maxime Saffon, Rafael Jiménez-Flores, Michel Britten, Yves Pouliot PII:
S0958-6946(15)00015-1
DOI:
10.1016/j.idairyj.2015.01.004
Reference:
INDA 3791
To appear in:
International Dairy Journal
Received Date: 28 September 2014 Revised Date:
7 January 2015
Accepted Date: 8 January 2015
Please cite this article as: Saffon, M., Jiménez-Flores, R., Britten, M., Pouliot, Y., Effect of heating whey proteins in the presence of milk fat globule membrane extract or phospholipids from buttermilk, International Dairy Journal (2015), doi: 10.1016/j.idairyj.2015.01.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effect of heating whey proteins in the presence of milk fat globule membrane extract or
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phospholipids from buttermilk
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Maxime Saffona, Rafael Jiménez-Floresb, Michel Brittenc, Yves Pouliota,*
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Comtois Building, Laval University, Quebec City, QC, G1V 0A6 Canada
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CA, 93407 USA
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QC, J2S 8E3 Canada
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Dairy Products Technology Center, California Polytechnic State University, San Luis Obispo,
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Food Research and Development Centre, Agriculture and Agri-Food Canada, St-Hyacinthe,
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STELA Dairy Research Centre, Institute of Nutrition and Functional Foods (INAF), Paul-
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* Corresponding author. Tel.: +1 418 656 5988
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E-mail address: [email protected] (Y. Pouliot)
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Abstract
27 The objective of this study was to determine the contribution of phospholipids from
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buttermilk as a nucleus in the heat-induced aggregation of whey proteins. Solutions of whey
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proteins (5%, w/v) were adjusted to pH 4.6 or 6.8 and then heated at 65 or 80 °C for 25 min with
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or without 1% (w/v) of milk fat globule membrane (MFGM) extract or phospholipid powder. The
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aggregation mechanisms were characterised using analysis with Ellman’s reagent, one-
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dimensional gel electrophoresis, thin-layer chromatography, and three-dimensional confocal
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laser-scanning microscopy. Three-dimensional images showed protein/phospholipid interactions
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in the presence of MFGM extract or phospholipids, and thin-layer chromatography plates showed
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no trace of free phospholipids after 20 min at pH 4.6. Overall, the results demonstrate that
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phospholipids from buttermilk were involved in the formation of protein aggregates through the
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MFGM fragments at a low temperature, whereas phospholipids could interact directly with the
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proteins at a higher temperature (80 °C).
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1.
Introduction
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Buttermilk is a unique product due to its concentration of milk fat globule membrane (MFGM) fragments and related materials (proteins, phospholipids, and sphingolipids) that have
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been associated with very promising health properties, ranging from antiviral to anticancer
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activities (Dewettinck et al., 2008). However, the isolation of the MFGM material is challenging,
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due to some interactions with other proteins during processing (Bédard-St-Amand, 2009; Morin,
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2006).
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A previous study showed that the addition of buttermilk constituents to whey protein
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concentrate prior to heat treatment induced the formation of protein aggregates with reduced
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water-holding capacity (Saffon, Britten, & Pouliot, 2011). Moreover, the latest results
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demonstrated that protein aggregates involving whey proteins, caseins, and MFGM proteins
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formed during the butter-making process (Saffon, Jiménez-Flores, Britten, & Pouliot, 2014). The
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pre-formed aggregates acted as an aggregation nucleus for whey proteins. The results of that
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study also suggested that phospholipids could also act as an aggregation nucleus.
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In bovine milk, most of the phospholipids (50-60%) are attached to the fat membrane
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(McPherson & Kitchen, 1983; Singh, 2006). Therefore, the presence of phospholipids in heat-
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induced aggregates is due mostly to interactions of the MFGM proteins with other proteins.
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However, Houlihan, Goddard, Nottingham, Kitchen, and Masters (1992) suggested that
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membrane lipids could also be involved in the heat-induced aggregation of the MFGM with
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proteins in whole milk. This idea is reinforced by the fact that phospholipids become more
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accessible after the pasteurisation of milk, as demonstrated by Morin, Jiménez-Flores, and
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Pouliot (2007).
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Interactions between milk proteins and phospholipids have been shown in model systems.
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The formation of the complex is initiated by ionic attraction between charged amino acid residues
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of the protein (i.e., lysine or arginine) and the polar head group of the lipid (i.e., phosphate or
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carboxylic group). Then, the complex is stabilised by hydrophobic interactions between
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hydrophobic residues of the protein (e.g., tryptophan) and the hydrocarbon chain of the lipid
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(Brown, Carroll, Pfeffer, & Sampugna, 1983; Dufourcq & Faucon, 1977). However,
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β-lactoglobulin in its native form is generally unable to form a complex with phospholipids or
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lipids in general. A certain degree of unfolding is necessary to expose the initially buried helical
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regions and hydrophobic groups (Bos & Nylander, 1996; Brown et al., 1983; Ong, Marchesi, &
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Prestegard, 1981). Spector and Fletcher (1970) concluded that β-lactoglobulin offers only one
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high-energy bonding site for long-chain lipids and one weak-energy bonding site. In contrast,
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bovine serum albumin offers six high-energy bonding sites.
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The objective of the present study was to determine if phospholipids from buttermilk interact with whey proteins during heat treatment. Whey proteins were heated in solution in the
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presence of MFGM extract from whey buttermilk or of phospholipids from regular buttermilk.
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The composition of the aggregates was characterised by determination of free thiol-group
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concentration, gel electrophoresis, thin-layer chromatography, and three-dimensional confocal
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laser-scanning microscopy.
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2.
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2.1.
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Materials and methods
Materials
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Whey cream (pasteurised at 82.2 °C for 35 s; milk pasteurised at 73.3 °C for 16 s) from Cheddar cheese production was donated by Hilmar Ingredients (Hilmar, CA, USA). Whey
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protein isolate (WPI) was obtained from Davisco Foods International (Eden Prairie, MN, USA),
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Phospholipid Concentrate 700 from Fonterra (Edgecumbe, New Zealand), Lissamine Rhodamine
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B from Avanti Polar Lipids (Alabaster, AL, USA), Fast Green FCF from Sigma Chemical
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Company (St. Louis, MO, USA), and DTNB (Ellman’s reagent) from Thermo Scientific
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(Rockford, IL, USA). All other reagents were from Fisher Scientific (Fair Lawn, NJ, USA).
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Isolation of milk fat globule membrane material
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Whey cream was processed at 12 °C using a continuous pilot-plant butter churn (Egli, Bütschwil, Switzerland). Immediately after churning, the whey buttermilk was filtered through
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cheesecloth. The filtered buttermilk was centrifuged at 50,000 × g for 120 min at 4 °C (Beckman
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L7-35 ultracentrifuge; Beckman Coulter, Indianapolis, IN, USA). The supernatant was removed,
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and the pellet was dissolved in PBS buffer in the presence of Triton X-100 (1%, w/v). The
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hydrated pellet was centrifuged at 50,000 × g for 120 min at 4 °C. The supernatant was removed,
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and the pellet was hydrated in water to a 10% (w/v) solution. The solution was mixed in a
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blender, sonicated for 1 min, and finally stored at −20 °C until analysis.
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WPI powder was dispersed in deionised water to a concentration of 5.0% (w/v) protein,
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2.3.
Heat treatment
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and the pH was adjusted to 4.6 with 1 M HCl or 6.8 with 1 M NaOH. The solutions were then
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dispersed overnight at 4 °C. The WPI solutions were heated from 8 to 65 °C or from 8 to 80 °C in 5
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a thermostatically controlled water bath maintained at 72 or 87 °C for a total time of 25 min. The
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solutions were constantly stirred using a helical laboratory mixer (model BDC250; Caframo,
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Georgian Bluffs, Canada) at medium speed during heating. The heated solutions were cooled
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down to room temperature. Whey protein solutions with the addition of 1 g of MFGM extract or
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phospholipid powder per 100 mL were also heated under the same conditions. The samples were
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named WPI for whey protein solution, WPI+M for whey proteins plus MFGM extract solution,
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and WPI+P for whey proteins plus phospholipid solution. All heating experiments were repeated
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three times.
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2.4.
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Concentration of free thiol groups
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The accessibility of free thiol groups was measured according to the method of Ellman
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(1959). Briefly, 250 µL samples of the mixtures were collected every 5 min during the heating
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time and mixed with 2.5 mL 0.1 M sodium phosphate buffer (containing 1 mM EDTA; pH 8.0)
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and 0.1 mL DTNB (Ellman’s reagent) solution (4 mg in 1 mL sodium phosphate buffer). The
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tubes were mixed by hand and then incubated at room temperature for 30 min. Absorbance was
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measured at 412 nm using a SpectraMax Plus spectrophotometer (Molecular Devices, Sunnyvale,
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CA, USA) after setting a blank. The absorbance of each sample was also measured without the
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addition of DTNB. The concentration of free thiol groups was measured in moles per gram of
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proteins (mol gprot-1).
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2.5.
Gel electrophoresis
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Protein profiles were determined using the sodium dodecyl sulphate polyacrylamide gel
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electrophoresis (SDS-PAGE) technique under non-reducing conditions according to the method
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of Laemmli (1970). Briefly, samples were taken every 5 min during the heating time and diluted
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1:10 in deionised water and then in alkaline buffer. Then, 25-µL samples were loaded onto 15%
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acrylamide SDS-PAGE gels. Analyses were conducted in a mini-Protean II system (Bio-Rad
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Laboratories, Hercules, CA, USA) at 90 V. The gels were stained with Coomassie Brilliant Blue
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(Sigma Chemical Company). Protein classes were determined according to their molecular
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weights by comparison with a molecular weight standard (Precision Plus All Blue Protein
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Standard; Bio-Rad Laboratories).
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2.6.
Thin-layer chromatography
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Samples of the WPI+P solution were taken every 5 min during the heating time and loaded (5 µL) directly onto a glass-backed silica thin-layer chromatography plate using a glass
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capillary. The plates were placed in a chamber filled with a polar solvent mixture containing
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65 mL chloroform, 25 mL methanol, and 4 mL deionised water. The plates were kept in the
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chamber for 60 to 90 min and then dried in a vacuum oven for 15 min. The dried plates were
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placed overnight in an iodine chamber with an iodine pellet.
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Samples of the mixtures were taken at 0, 15, and 25 min during the heating time and
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2.7.
Confocal laser-scanning microscopy
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diluted 1:2 in deionised water. Then, 50 µL of the diluted samples was dyed with 2 µL Lissamine
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Rhodamine B and 2 µL Fast Green FCF. The samples were mixed and stored in the dark for 7
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20 min. A 25 µL aliquot of the dyed solution was deposited onto a slide and fixed with agarose
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(50 µL; 0.5%, w/v, in deionised water). Three-dimensional images were taken by scanning the
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sample across a defined section along the z-axis using an Olympus Fluoview FV1000 inverted
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confocal laser-scanning microscope (Olympus America, Center Valley, PA, USA).
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2.8.
Statistical analysis
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All heat treatments and experiments were performed in triplicate. The independent variables were composition (control protein dispersion and dispersions with added MFGM or
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phospholipids), time (holding time in the water bath), temperature (heating temperature), and pH
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(pH of the solutions during heating). The concentration of exposed thiol groups was studied in a
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3*2*2*2 factorial experiment. Statistical analysis was carried out with the SAS software program
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(SAS Institute, Cary, NC, USA) using the PROC GLM procedure. The results were considered
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significantly different when P < 0.05.
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Results and discussion
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3.1.
Effect of heating whey proteins in the presence of milk fat globule membrane extract and
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phospholipids
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As shown in Fig. 1, visual differences were observed between pellets from the
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sedimentation of mixtures following the heat treatment. At pH 4.6, a more compact pellet was
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obtained with WPI than with WPI+M or WPI+P. The WPI pellet had a harder texture (similar to
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a gel) than did the pellets from the other mixtures, which were softer and easier to disturb by 8
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gently shaking the tubes. No pellets were observed at pH 6.8 for both temperatures, suggesting
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that the formation of soluble and non-sedimentable aggregates was promoted at this pH level.
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These observations are in agreement with the previous results indicating that the presence of
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buttermilk constituents controlled the size and number of particles formed during heating by
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offering more sites for aggregation (Saffon et al., 2014).
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The statistical analysis of free thiol values following the heating of whey and the mixtures showed that only the composition*temperature (P = 0.0094) and temperature*pH (P = 0.0049)
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interactions were significant with regard to the concentration of free thiol groups. These
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observations support the view that the accessibility of free thiol groups depends on the
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temperature and pH. It has been known since Pantaloni (1964) that exposure of the free thiol
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group of β-lactoglobulin (Cys121) starts at 70 °C, although in a low proportion (6%), and indicates
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the irreversible conformational change of the proteins. However, β-lactoglobulin is irreversibly
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unfolded at a temperature close to 78.5 °C, making the free thiol groups more accessible for
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thiol/disulphide exchange reactions (Schokker, Singh, Pinder, Norris, & Creamer, 1999). Raising
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the temperature above 80 °C increased the liberation of free thiol groups. Hoffmann and van Mil
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(1997) demonstrated that the aggregation of β-lactoglobulin is related to the pH-dependent
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reactivity of the free thiol group. Their results showed that the accessibility of the free thiol group
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of the protein is better at pH 8.0 than at pH 7.0, because the pK value of the thiol group is around
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8.2. However, the presence of MFGM extract or phospholipids affected the denaturation of
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β-lactoglobulin in some way.
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3.2.
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phospholipids
Changes in protein denaturation in the presence of milk fat globule membrane extract and
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Changes in the protein profiles of the samples as a function of heating time (80 °C) are presented in Fig. 2. At pH 6.8, the contents of native β-lactoglobulin and α-lactalbumin decreased
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after 15 min of heating (until the end) in WPI+M but after only 10 min in WPI. This is in
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agreement with previously reported results that the presence of buttermilk constituents delayed
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the loss of native β-lactoglobulin and α-lactalbumin (Saffon et al., 2014). The decreasing amount
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of native whey proteins at each heating time was correlated with the aggregation process.
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Under non-reducing conditions, only butyrophilin (BTN: 67 kDa), and Cluster of
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Differentiation 36 or Periodic Acid Schiff IV (CD36 or PAS IV: 76–78 kDa) were detected in the
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mixtures. Ye, Singh, Taylor, and Anema (2002) reported that MFGM proteins are more heat-
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sensitive than whey proteins are. The pasteurisation treatment applied to the whey or whey cream
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used for this study may have been sufficient to denature most of the MFGM proteins. Results
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obtained by Ye et al. (2002) also suggested that xanthine dehydrogenase/oxidase (XDH/XO:
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150 kDa) and butyrophilin form a protein complex with a higher molecular weight during heating
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above 60 °C. This complex could be the band located around 250 kDa (Fig. 2). No decrease in
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the amount of the native whey proteins or MFGM proteins was observed at pH 4.6 or during
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heating at 65 °C (for both pH levels). The changes in the protein profile of the WPI+P mixture
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did not differ from the changes in the profile of WPI, except that the loss of the native forms of
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β-lactoglobulin and α-lactalbumin was delayed by 5 min (Fig. 2).
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3.3.
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membrane extract or phospholipids
Microstructure of the aggregates upon heating in the presence of milk fat globule
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Despite the similar protein profile changes, the microstructure of the aggregates was
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different in the presence of MFGM fragments (Fig. 3). In WPI, non-soluble aggregates were 10
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composed only of proteins (green). Phospholipids (red) were present in very small amounts and
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were not integrated into the structure of the protein aggregates. Some protein/phospholipid or
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protein/MFGM protein interactions could be observed, but they were isolated and limited. In the
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presence of MFGM material (Fig. 4), MFGM fragments or phospholipids were part of the protein
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aggregate structures at pH 4.6, even after only 15 min of heating at 80 °C. Most of the red dots
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were located on the side of the aggregates, but some were also buried inside their structure.
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Pictures were taken from different angles in order to confirm that the red dots and green dots
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were connected. According to Ye, Singh, Oldfield, and Anema (2004) and Ye, Singh, Taylor, and
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Anema (2004), interactions between whey proteins and MFGM proteins occur because of the
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exposure of free thiol groups of MFGM proteins during the initial stage of whey protein
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denaturation and involve mainly thiol/disulphide exchange reactions.
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As shown in Fig. 5, phospholipids (red dots) were integrated into the structure of the protein aggregates (green) at pH 4.6 during heating at 65 °C only. Phospholipids were present on
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the side or buried in the structure of the protein aggregates. The authors hypothesise that
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phospholipids could act as an aggregation nucleus during the heat-induced denaturation of whey
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proteins. Most protein aggregation mechanisms (including for β-lactoglobulin) are defined as
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being nucleation-dependent and are therefore initiated by the formation of an aggregation nucleus
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(Wang, Nema, & Teagarden, 2010). Later in the aggregation process, non-native monomers,
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denatured proteins, and/or small aggregates are incorporated into the nucleus to form larger
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aggregates. Wang et al. (2010) defined the last state of protein denaturation as the precipitation
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(loss of solubility) of aggregates. This step was not reached at pH 6.8. Only a few isolated non-
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soluble particles were observed.
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3.4.
Effect of heating time on free phospholipids in heated mixtures
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Changes in “free” phospholipids as a function of heating time are presented in Fig. 6. It can be observed that free phospholipids started decreasing after 25 min of heating at pH 6.8
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(slight decrease) and after 20 min at pH 4.6 (complete loss). A longer time than expected was
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necessary to bind a large amount of phospholipids to whey proteins at 80 °C. Interactions
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between phospholipids and whey proteins during the early stage of heating still appeared but
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involved only a small proportion of phospholipids or were weak interactions (easily breakable by
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the thin-layer chromatography solvents). The thin-layer chromatography plates used in this study
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did not allow identification of all spots, but the phospholipid powder had been analysed
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beforehand by high-performance liquid chromatography.
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The following components were detected: neutral lipids, lactosylceramide,
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sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and
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phosphatidylserine. Cornell and Patterson (1989) reported interactions between β-lactoglobulin
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and phospholipids at pH 4.4. Their results are consistent with the idea that positively charged
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groups of the proteins interact with negatively charged lipids. At pH 4.6, β-lactoglobulin has a
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positive net charge, phosphatidylcholine and phosphatidylethanolamine are isoelectric (other
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phospholipids are ionised at pH > 4.0) (Hauser & Phillips, 1979). As described by Cornell and
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Patterson (1989), an electrostatic attraction between phosphatidylcholine and β-lactoglobulin
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could be expected. Earlier, Brown, Carroll, Pfeffer, and Sampugna (1983) also reported an
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interaction between phosphatidylcholine and β-lactoglobulin involving both ionic and
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hydrophobic interactions after treatment with a helix-forming solvent. Ionic interactions make it
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possible to position the lipid and protein molecules, and then hydrophobic interactions stabilise
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the complex. Later, Lefèvre and Subirade (1999, 2000) used Fourier transform infrared
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spectroscopy to better understand interactions between β-lactoglobulin and phospholipids. Those
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studies revealed hydrophobic interactions between the protein and sphingomyelin as well as
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electrostatic interactions with phosphatidylserine.
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The present study shows that the presence of MFGM fragments or phospholipids delayed
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the heat-induced denaturation of whey proteins but did not affect the exposure of free thiol
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groups of the whey proteins. However, the addition of MFGM extract or phospholipids clearly
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modified the microstructure of the particles formed after heating. Three-dimensional confocal
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laser-scanning micrographs confirmed the presence of phospholipids buried in the structure of the
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protein aggregates at pH 4.6. However, only a small fraction of phospholipids were bound during
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the early stage of denaturation, or the interactions were easily breakable. A longer heating time
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(>15 min at pH 4.6 or >20 min at pH 6.8) was required to strongly bind phospholipids to whey
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proteins at 80 °C. It appears that phospholipids were incorporated into the structure of protein
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aggregates through MFGM fragments at a lower temperature (65 °C), whereas phospholipids
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could interact directly with the proteins at a higher temperature (80 °C).
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Acknowledgements
This work was funded by the Fonds québécois de la recherche sur la nature et les
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technologies (FQRNT; Quebec City, QC, Canada), Novalait Inc. (Ste-Foy, QC, Canada), and the
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Ministère de l’Agriculture, des Pêcheries et de l’Alimentation du Québec (MAPAQ; Quebec
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City, QC, Canada). 13
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Biochemistry, 20, 4283–4292. Pantaloni, D. (1964). Etude de la transformation R→S de la beta-lactoglobuline par
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spectropolarimétrie et par spectrophotométrie de différences. Comptes Rendus de
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l’Académie des Sciences, 258, 5753–5756.
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Saffon, M., Britten, M., & Pouliot, Y. (2011). Thermal aggregation of whey proteins in the presence of buttermilk concentrate. Journal of Food Engineering, 103, 244–250. Saffon, M., Jiménez-Flores, R., Britten, M., & Pouliot, Y. (2014). On the use of buttermilk
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components as aggregation nuclei during the heat-induced denaturation of whey proteins.
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Journal of Food Engineering, 132, 21–28.
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Schokker, E. P., Singh, H., Pinder, D. N., Norris, G. E., & Creamer, L. K. (1999). Characterization of intermediates formed during heat-induced aggregation of β-
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lactoglobulin AB at neutral pH. International Dairy Journal, 9, 791–800.
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Current Opinion in Colloid and Interface Science, 11, 154–163.
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Singh, H. (2006). The milk fat globule membrane—A biophysical system for food applications.
Spector, A. A., & Fletcher, J. E. (1970). Binding of long chain fatty acids to β-lactoglobulin. Lipids, 5, 403–411.
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Wang, W., Nema, S., & Teagarden, D. (2010). Protein aggregation—Pathways and influencing factors. International Journal of Pharmaceutics, 390, 89–99.
Ye, A., Singh, H., Oldfield, D. J., & Anema, S. (2004). Kinetics of heat-induced association of β-
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lactoglobulin and α-lactalbumin with milk fat globule membrane in whole milk.
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International Dairy Journal, 14, 389–398.
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Ye, A., Singh, H., Taylor, M. W., & Anema, S. (2002). Characterization of protein components
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of natural and heat-treated milk fat globule membranes. International Dairy Journal, 12,
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fat globule membrane proteins during heat treatment of whole milk. Lait, 84, 269–283.
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Ye, A., Singh, H., Taylor, M. W., & Anema, S. (2004). Interactions of whey proteins with milk
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Fig. 1. Pictures of heated whey protein isolate (a), whey protein isolate plus milk fat globule membrane extract (b), and whey protein isolate plus phospholipids (c) at pH 4.6 and 80 °C
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after 1 h of incubation in an ice water bath.
Fig. 2. Evolution of the protein profiles of the different mixtures (WPI: whey protein isolate;
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WPI+M: whey protein isolate plus milk fat globule membrane extract; WPI+P: whey protein isolate plus phospholipids) after 0 min (a), 10 min (b), 15 min (c), and 20 min (d) of heating
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Fig. 3. Three-dimensional confocal laser-scanning microscope pictures of whey protein isolate heated for 25 min at 80 °C and pH 4.6.
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Fig. 4. Three-dimensional confocal laser-scanning microscope pictures of whey protein
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isolate plus milk fat globule membrane extract heated for 15 min at 80 °C and pH 4.6.
Fig. 5. Three-dimensional confocal laser-scanning microscope pictures of whey protein isolate plus phospholipids heated for 15 min at 65 °C and pH 4.6.
Fig. 6. Changes in the phospholipids profiles of whey protein isolate plus phospholipids after 0 min (a), 10 min (b), 15 min (c), 20 min (d), and 25 min (e) of heating at 80 °C. The picture was converted to negative mode (right panel) to make the spots easier to see.
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S0958-6946(15)00015-1
DOI:
10.1016/j.idairyj.2015.01.004
Reference:
INDA 3791
To appear in:
International Dairy Journal
Received Date: 28 September 2014 Revised Date:
7 January 2015
Accepted Date: 8 January 2015
Please cite this article as: Saffon, M., Jiménez-Flores, R., Britten, M., Pouliot, Y., Effect of heating whey proteins in the presence of milk fat globule membrane extract or phospholipids from buttermilk, International Dairy Journal (2015), doi: 10.1016/j.idairyj.2015.01.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effect of heating whey proteins in the presence of milk fat globule membrane extract or
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phospholipids from buttermilk
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Maxime Saffona, Rafael Jiménez-Floresb, Michel Brittenc, Yves Pouliota,*
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a
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Comtois Building, Laval University, Quebec City, QC, G1V 0A6 Canada
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b
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CA, 93407 USA
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c
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QC, J2S 8E3 Canada
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Dairy Products Technology Center, California Polytechnic State University, San Luis Obispo,
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Food Research and Development Centre, Agriculture and Agri-Food Canada, St-Hyacinthe,
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STELA Dairy Research Centre, Institute of Nutrition and Functional Foods (INAF), Paul-
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* Corresponding author. Tel.: +1 418 656 5988
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E-mail address: [email protected] (Y. Pouliot)
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Abstract
27 The objective of this study was to determine the contribution of phospholipids from
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buttermilk as a nucleus in the heat-induced aggregation of whey proteins. Solutions of whey
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proteins (5%, w/v) were adjusted to pH 4.6 or 6.8 and then heated at 65 or 80 °C for 25 min with
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or without 1% (w/v) of milk fat globule membrane (MFGM) extract or phospholipid powder. The
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aggregation mechanisms were characterised using analysis with Ellman’s reagent, one-
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dimensional gel electrophoresis, thin-layer chromatography, and three-dimensional confocal
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laser-scanning microscopy. Three-dimensional images showed protein/phospholipid interactions
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in the presence of MFGM extract or phospholipids, and thin-layer chromatography plates showed
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no trace of free phospholipids after 20 min at pH 4.6. Overall, the results demonstrate that
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phospholipids from buttermilk were involved in the formation of protein aggregates through the
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MFGM fragments at a low temperature, whereas phospholipids could interact directly with the
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proteins at a higher temperature (80 °C).
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1.
Introduction
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Buttermilk is a unique product due to its concentration of milk fat globule membrane (MFGM) fragments and related materials (proteins, phospholipids, and sphingolipids) that have
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been associated with very promising health properties, ranging from antiviral to anticancer
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activities (Dewettinck et al., 2008). However, the isolation of the MFGM material is challenging,
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due to some interactions with other proteins during processing (Bédard-St-Amand, 2009; Morin,
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2006).
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A previous study showed that the addition of buttermilk constituents to whey protein
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concentrate prior to heat treatment induced the formation of protein aggregates with reduced
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water-holding capacity (Saffon, Britten, & Pouliot, 2011). Moreover, the latest results
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demonstrated that protein aggregates involving whey proteins, caseins, and MFGM proteins
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formed during the butter-making process (Saffon, Jiménez-Flores, Britten, & Pouliot, 2014). The
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pre-formed aggregates acted as an aggregation nucleus for whey proteins. The results of that
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study also suggested that phospholipids could also act as an aggregation nucleus.
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In bovine milk, most of the phospholipids (50-60%) are attached to the fat membrane
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(McPherson & Kitchen, 1983; Singh, 2006). Therefore, the presence of phospholipids in heat-
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induced aggregates is due mostly to interactions of the MFGM proteins with other proteins.
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However, Houlihan, Goddard, Nottingham, Kitchen, and Masters (1992) suggested that
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membrane lipids could also be involved in the heat-induced aggregation of the MFGM with
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proteins in whole milk. This idea is reinforced by the fact that phospholipids become more
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accessible after the pasteurisation of milk, as demonstrated by Morin, Jiménez-Flores, and
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Pouliot (2007).
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Interactions between milk proteins and phospholipids have been shown in model systems.
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The formation of the complex is initiated by ionic attraction between charged amino acid residues
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of the protein (i.e., lysine or arginine) and the polar head group of the lipid (i.e., phosphate or
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carboxylic group). Then, the complex is stabilised by hydrophobic interactions between
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hydrophobic residues of the protein (e.g., tryptophan) and the hydrocarbon chain of the lipid
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(Brown, Carroll, Pfeffer, & Sampugna, 1983; Dufourcq & Faucon, 1977). However,
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β-lactoglobulin in its native form is generally unable to form a complex with phospholipids or
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lipids in general. A certain degree of unfolding is necessary to expose the initially buried helical
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regions and hydrophobic groups (Bos & Nylander, 1996; Brown et al., 1983; Ong, Marchesi, &
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Prestegard, 1981). Spector and Fletcher (1970) concluded that β-lactoglobulin offers only one
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high-energy bonding site for long-chain lipids and one weak-energy bonding site. In contrast,
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bovine serum albumin offers six high-energy bonding sites.
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The objective of the present study was to determine if phospholipids from buttermilk interact with whey proteins during heat treatment. Whey proteins were heated in solution in the
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presence of MFGM extract from whey buttermilk or of phospholipids from regular buttermilk.
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The composition of the aggregates was characterised by determination of free thiol-group
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concentration, gel electrophoresis, thin-layer chromatography, and three-dimensional confocal
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laser-scanning microscopy.
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2.
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2.1.
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Materials and methods
Materials
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Whey cream (pasteurised at 82.2 °C for 35 s; milk pasteurised at 73.3 °C for 16 s) from Cheddar cheese production was donated by Hilmar Ingredients (Hilmar, CA, USA). Whey
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protein isolate (WPI) was obtained from Davisco Foods International (Eden Prairie, MN, USA),
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Phospholipid Concentrate 700 from Fonterra (Edgecumbe, New Zealand), Lissamine Rhodamine
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B from Avanti Polar Lipids (Alabaster, AL, USA), Fast Green FCF from Sigma Chemical
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Company (St. Louis, MO, USA), and DTNB (Ellman’s reagent) from Thermo Scientific
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(Rockford, IL, USA). All other reagents were from Fisher Scientific (Fair Lawn, NJ, USA).
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Isolation of milk fat globule membrane material
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Whey cream was processed at 12 °C using a continuous pilot-plant butter churn (Egli, Bütschwil, Switzerland). Immediately after churning, the whey buttermilk was filtered through
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cheesecloth. The filtered buttermilk was centrifuged at 50,000 × g for 120 min at 4 °C (Beckman
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L7-35 ultracentrifuge; Beckman Coulter, Indianapolis, IN, USA). The supernatant was removed,
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and the pellet was dissolved in PBS buffer in the presence of Triton X-100 (1%, w/v). The
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hydrated pellet was centrifuged at 50,000 × g for 120 min at 4 °C. The supernatant was removed,
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and the pellet was hydrated in water to a 10% (w/v) solution. The solution was mixed in a
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blender, sonicated for 1 min, and finally stored at −20 °C until analysis.
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WPI powder was dispersed in deionised water to a concentration of 5.0% (w/v) protein,
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2.3.
Heat treatment
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and the pH was adjusted to 4.6 with 1 M HCl or 6.8 with 1 M NaOH. The solutions were then
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dispersed overnight at 4 °C. The WPI solutions were heated from 8 to 65 °C or from 8 to 80 °C in 5
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a thermostatically controlled water bath maintained at 72 or 87 °C for a total time of 25 min. The
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solutions were constantly stirred using a helical laboratory mixer (model BDC250; Caframo,
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Georgian Bluffs, Canada) at medium speed during heating. The heated solutions were cooled
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down to room temperature. Whey protein solutions with the addition of 1 g of MFGM extract or
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phospholipid powder per 100 mL were also heated under the same conditions. The samples were
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named WPI for whey protein solution, WPI+M for whey proteins plus MFGM extract solution,
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and WPI+P for whey proteins plus phospholipid solution. All heating experiments were repeated
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three times.
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Concentration of free thiol groups
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The accessibility of free thiol groups was measured according to the method of Ellman
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(1959). Briefly, 250 µL samples of the mixtures were collected every 5 min during the heating
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time and mixed with 2.5 mL 0.1 M sodium phosphate buffer (containing 1 mM EDTA; pH 8.0)
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and 0.1 mL DTNB (Ellman’s reagent) solution (4 mg in 1 mL sodium phosphate buffer). The
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tubes were mixed by hand and then incubated at room temperature for 30 min. Absorbance was
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measured at 412 nm using a SpectraMax Plus spectrophotometer (Molecular Devices, Sunnyvale,
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CA, USA) after setting a blank. The absorbance of each sample was also measured without the
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addition of DTNB. The concentration of free thiol groups was measured in moles per gram of
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proteins (mol gprot-1).
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2.5.
Gel electrophoresis
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Protein profiles were determined using the sodium dodecyl sulphate polyacrylamide gel
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electrophoresis (SDS-PAGE) technique under non-reducing conditions according to the method
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of Laemmli (1970). Briefly, samples were taken every 5 min during the heating time and diluted
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1:10 in deionised water and then in alkaline buffer. Then, 25-µL samples were loaded onto 15%
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acrylamide SDS-PAGE gels. Analyses were conducted in a mini-Protean II system (Bio-Rad
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Laboratories, Hercules, CA, USA) at 90 V. The gels were stained with Coomassie Brilliant Blue
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(Sigma Chemical Company). Protein classes were determined according to their molecular
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weights by comparison with a molecular weight standard (Precision Plus All Blue Protein
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Standard; Bio-Rad Laboratories).
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2.6.
Thin-layer chromatography
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Samples of the WPI+P solution were taken every 5 min during the heating time and loaded (5 µL) directly onto a glass-backed silica thin-layer chromatography plate using a glass
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capillary. The plates were placed in a chamber filled with a polar solvent mixture containing
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65 mL chloroform, 25 mL methanol, and 4 mL deionised water. The plates were kept in the
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chamber for 60 to 90 min and then dried in a vacuum oven for 15 min. The dried plates were
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placed overnight in an iodine chamber with an iodine pellet.
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Samples of the mixtures were taken at 0, 15, and 25 min during the heating time and
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2.7.
Confocal laser-scanning microscopy
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diluted 1:2 in deionised water. Then, 50 µL of the diluted samples was dyed with 2 µL Lissamine
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Rhodamine B and 2 µL Fast Green FCF. The samples were mixed and stored in the dark for 7
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20 min. A 25 µL aliquot of the dyed solution was deposited onto a slide and fixed with agarose
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(50 µL; 0.5%, w/v, in deionised water). Three-dimensional images were taken by scanning the
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sample across a defined section along the z-axis using an Olympus Fluoview FV1000 inverted
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confocal laser-scanning microscope (Olympus America, Center Valley, PA, USA).
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2.8.
Statistical analysis
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All heat treatments and experiments were performed in triplicate. The independent variables were composition (control protein dispersion and dispersions with added MFGM or
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phospholipids), time (holding time in the water bath), temperature (heating temperature), and pH
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(pH of the solutions during heating). The concentration of exposed thiol groups was studied in a
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3*2*2*2 factorial experiment. Statistical analysis was carried out with the SAS software program
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(SAS Institute, Cary, NC, USA) using the PROC GLM procedure. The results were considered
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significantly different when P < 0.05.
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Results and discussion
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3.1.
Effect of heating whey proteins in the presence of milk fat globule membrane extract and
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phospholipids
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As shown in Fig. 1, visual differences were observed between pellets from the
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sedimentation of mixtures following the heat treatment. At pH 4.6, a more compact pellet was
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obtained with WPI than with WPI+M or WPI+P. The WPI pellet had a harder texture (similar to
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a gel) than did the pellets from the other mixtures, which were softer and easier to disturb by 8
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gently shaking the tubes. No pellets were observed at pH 6.8 for both temperatures, suggesting
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that the formation of soluble and non-sedimentable aggregates was promoted at this pH level.
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These observations are in agreement with the previous results indicating that the presence of
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buttermilk constituents controlled the size and number of particles formed during heating by
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offering more sites for aggregation (Saffon et al., 2014).
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The statistical analysis of free thiol values following the heating of whey and the mixtures showed that only the composition*temperature (P = 0.0094) and temperature*pH (P = 0.0049)
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interactions were significant with regard to the concentration of free thiol groups. These
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observations support the view that the accessibility of free thiol groups depends on the
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temperature and pH. It has been known since Pantaloni (1964) that exposure of the free thiol
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group of β-lactoglobulin (Cys121) starts at 70 °C, although in a low proportion (6%), and indicates
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the irreversible conformational change of the proteins. However, β-lactoglobulin is irreversibly
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unfolded at a temperature close to 78.5 °C, making the free thiol groups more accessible for
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thiol/disulphide exchange reactions (Schokker, Singh, Pinder, Norris, & Creamer, 1999). Raising
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the temperature above 80 °C increased the liberation of free thiol groups. Hoffmann and van Mil
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(1997) demonstrated that the aggregation of β-lactoglobulin is related to the pH-dependent
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reactivity of the free thiol group. Their results showed that the accessibility of the free thiol group
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of the protein is better at pH 8.0 than at pH 7.0, because the pK value of the thiol group is around
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8.2. However, the presence of MFGM extract or phospholipids affected the denaturation of
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β-lactoglobulin in some way.
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3.2.
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phospholipids
Changes in protein denaturation in the presence of milk fat globule membrane extract and
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Changes in the protein profiles of the samples as a function of heating time (80 °C) are presented in Fig. 2. At pH 6.8, the contents of native β-lactoglobulin and α-lactalbumin decreased
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after 15 min of heating (until the end) in WPI+M but after only 10 min in WPI. This is in
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agreement with previously reported results that the presence of buttermilk constituents delayed
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the loss of native β-lactoglobulin and α-lactalbumin (Saffon et al., 2014). The decreasing amount
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of native whey proteins at each heating time was correlated with the aggregation process.
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Under non-reducing conditions, only butyrophilin (BTN: 67 kDa), and Cluster of
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Differentiation 36 or Periodic Acid Schiff IV (CD36 or PAS IV: 76–78 kDa) were detected in the
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mixtures. Ye, Singh, Taylor, and Anema (2002) reported that MFGM proteins are more heat-
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sensitive than whey proteins are. The pasteurisation treatment applied to the whey or whey cream
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used for this study may have been sufficient to denature most of the MFGM proteins. Results
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obtained by Ye et al. (2002) also suggested that xanthine dehydrogenase/oxidase (XDH/XO:
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150 kDa) and butyrophilin form a protein complex with a higher molecular weight during heating
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above 60 °C. This complex could be the band located around 250 kDa (Fig. 2). No decrease in
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the amount of the native whey proteins or MFGM proteins was observed at pH 4.6 or during
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heating at 65 °C (for both pH levels). The changes in the protein profile of the WPI+P mixture
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did not differ from the changes in the profile of WPI, except that the loss of the native forms of
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β-lactoglobulin and α-lactalbumin was delayed by 5 min (Fig. 2).
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membrane extract or phospholipids
Microstructure of the aggregates upon heating in the presence of milk fat globule
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Despite the similar protein profile changes, the microstructure of the aggregates was
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different in the presence of MFGM fragments (Fig. 3). In WPI, non-soluble aggregates were 10
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composed only of proteins (green). Phospholipids (red) were present in very small amounts and
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were not integrated into the structure of the protein aggregates. Some protein/phospholipid or
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protein/MFGM protein interactions could be observed, but they were isolated and limited. In the
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presence of MFGM material (Fig. 4), MFGM fragments or phospholipids were part of the protein
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aggregate structures at pH 4.6, even after only 15 min of heating at 80 °C. Most of the red dots
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were located on the side of the aggregates, but some were also buried inside their structure.
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Pictures were taken from different angles in order to confirm that the red dots and green dots
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were connected. According to Ye, Singh, Oldfield, and Anema (2004) and Ye, Singh, Taylor, and
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Anema (2004), interactions between whey proteins and MFGM proteins occur because of the
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exposure of free thiol groups of MFGM proteins during the initial stage of whey protein
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denaturation and involve mainly thiol/disulphide exchange reactions.
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As shown in Fig. 5, phospholipids (red dots) were integrated into the structure of the protein aggregates (green) at pH 4.6 during heating at 65 °C only. Phospholipids were present on
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the side or buried in the structure of the protein aggregates. The authors hypothesise that
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phospholipids could act as an aggregation nucleus during the heat-induced denaturation of whey
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proteins. Most protein aggregation mechanisms (including for β-lactoglobulin) are defined as
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being nucleation-dependent and are therefore initiated by the formation of an aggregation nucleus
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(Wang, Nema, & Teagarden, 2010). Later in the aggregation process, non-native monomers,
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denatured proteins, and/or small aggregates are incorporated into the nucleus to form larger
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aggregates. Wang et al. (2010) defined the last state of protein denaturation as the precipitation
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(loss of solubility) of aggregates. This step was not reached at pH 6.8. Only a few isolated non-
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soluble particles were observed.
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3.4.
Effect of heating time on free phospholipids in heated mixtures
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Changes in “free” phospholipids as a function of heating time are presented in Fig. 6. It can be observed that free phospholipids started decreasing after 25 min of heating at pH 6.8
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(slight decrease) and after 20 min at pH 4.6 (complete loss). A longer time than expected was
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necessary to bind a large amount of phospholipids to whey proteins at 80 °C. Interactions
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between phospholipids and whey proteins during the early stage of heating still appeared but
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involved only a small proportion of phospholipids or were weak interactions (easily breakable by
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the thin-layer chromatography solvents). The thin-layer chromatography plates used in this study
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did not allow identification of all spots, but the phospholipid powder had been analysed
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beforehand by high-performance liquid chromatography.
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The following components were detected: neutral lipids, lactosylceramide,
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sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and
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phosphatidylserine. Cornell and Patterson (1989) reported interactions between β-lactoglobulin
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and phospholipids at pH 4.4. Their results are consistent with the idea that positively charged
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groups of the proteins interact with negatively charged lipids. At pH 4.6, β-lactoglobulin has a
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positive net charge, phosphatidylcholine and phosphatidylethanolamine are isoelectric (other
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phospholipids are ionised at pH > 4.0) (Hauser & Phillips, 1979). As described by Cornell and
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Patterson (1989), an electrostatic attraction between phosphatidylcholine and β-lactoglobulin
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could be expected. Earlier, Brown, Carroll, Pfeffer, and Sampugna (1983) also reported an
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interaction between phosphatidylcholine and β-lactoglobulin involving both ionic and
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hydrophobic interactions after treatment with a helix-forming solvent. Ionic interactions make it
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possible to position the lipid and protein molecules, and then hydrophobic interactions stabilise
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the complex. Later, Lefèvre and Subirade (1999, 2000) used Fourier transform infrared
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spectroscopy to better understand interactions between β-lactoglobulin and phospholipids. Those
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studies revealed hydrophobic interactions between the protein and sphingomyelin as well as
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electrostatic interactions with phosphatidylserine.
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The present study shows that the presence of MFGM fragments or phospholipids delayed
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the heat-induced denaturation of whey proteins but did not affect the exposure of free thiol
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groups of the whey proteins. However, the addition of MFGM extract or phospholipids clearly
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modified the microstructure of the particles formed after heating. Three-dimensional confocal
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laser-scanning micrographs confirmed the presence of phospholipids buried in the structure of the
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protein aggregates at pH 4.6. However, only a small fraction of phospholipids were bound during
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the early stage of denaturation, or the interactions were easily breakable. A longer heating time
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(>15 min at pH 4.6 or >20 min at pH 6.8) was required to strongly bind phospholipids to whey
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proteins at 80 °C. It appears that phospholipids were incorporated into the structure of protein
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aggregates through MFGM fragments at a lower temperature (65 °C), whereas phospholipids
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could interact directly with the proteins at a higher temperature (80 °C).
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Acknowledgements
This work was funded by the Fonds québécois de la recherche sur la nature et les
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technologies (FQRNT; Quebec City, QC, Canada), Novalait Inc. (Ste-Foy, QC, Canada), and the
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Ministère de l’Agriculture, des Pêcheries et de l’Alimentation du Québec (MAPAQ; Quebec
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City, QC, Canada). 13
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Fig. 1. Pictures of heated whey protein isolate (a), whey protein isolate plus milk fat globule membrane extract (b), and whey protein isolate plus phospholipids (c) at pH 4.6 and 80 °C
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after 1 h of incubation in an ice water bath.
Fig. 2. Evolution of the protein profiles of the different mixtures (WPI: whey protein isolate;
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WPI+M: whey protein isolate plus milk fat globule membrane extract; WPI+P: whey protein isolate plus phospholipids) after 0 min (a), 10 min (b), 15 min (c), and 20 min (d) of heating
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at 80 °C using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) under non-reducing conditions. Letters a to d correspond to samples at pH 6.8, and letters a’ to d’ to pH 4.6.
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Fig. 3. Three-dimensional confocal laser-scanning microscope pictures of whey protein isolate heated for 25 min at 80 °C and pH 4.6.
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Fig. 4. Three-dimensional confocal laser-scanning microscope pictures of whey protein
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isolate plus milk fat globule membrane extract heated for 15 min at 80 °C and pH 4.6.
Fig. 5. Three-dimensional confocal laser-scanning microscope pictures of whey protein isolate plus phospholipids heated for 15 min at 65 °C and pH 4.6.
Fig. 6. Changes in the phospholipids profiles of whey protein isolate plus phospholipids after 0 min (a), 10 min (b), 15 min (c), 20 min (d), and 25 min (e) of heating at 80 °C. The picture was converted to negative mode (right panel) to make the spots easier to see.
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