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Desfosforilación de Caseínas
Desfosforilación de Caseínas
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
A R T I C LE I N FO A B S T R A C T
Keywords: This study investigated the effects of dephosphorylation and sodium hexametaphosphate (SHMP) salt addition
Milk protein concentrate on the viscosity of milk protein concentrate (MPC) solutions. Dephosphorylation (DP) of casein was performed
Micellar casein using bovine alkaline phosphatase. Nuclear magnetic resonance (NMR) spectra showed that dephosphorylation
Sodium hexametaphosphate depleted the casein-bound phosphate region (CNP). SHMP addition (5 mM) had no impact on the 31P NMR
Dephosphorylation
spectra of DP-MPC; addition of 5 mM SHMP to control MPC (C-MPC) resulted in a shift in peaks associated with
Nuclear magnetic resonance
Gelation
the CNP region, possibly caused by SHMP sequestering calcium, leading to swelling of micelles. DP-MPC ex-
Rheology hibited a lower viscosity compared to C-MPC, with SHMP addition at 12.5 and 25 mM causing gelation of C-MPC
and DP-MPC solutions. This work confirmed the role that phosphate residues have in maintaining micelle
structural stability and provides new insights into controlling viscosity of MPC solutions.
1. Introduction solubility. McCarthy et al. (2017) showed that the addition of calcium
chelating salts to MPC solutions can increase protein solubility, but that
Milk protein concentrate (MPC) powders are produced using se- there is a significant concomitant increase in viscosity. The structural
paration processes such as ultrafiltration (UF) and diafiltration (DF) of stability of casein micelles is dependent on a delicate balance of at-
pasteurised skim milk. They have protein contents ranging from 40 to tractive (e.g., hydrophobic interactions) and repulsive (e.g., electro-
80% (w/w) (Havea, 2006, Sikand, Tong, Roy, Rodriguez-Saona, & static repulsion) forces between the constituent proteins. The colloidal
Murray, 2011) and a similar casein to whey protein ratio as the original calcium phosphate (CCP) associated with the proteins in casein micelles
skim milk (i.e., ∼80:20). After membrane filtration, the liquid MPC via phosphoserine residues can be depleted using approaches such as
may be heat treated and evaporated before spray drying, and while the ion-exchange and mineral chelation, thereby reducing micelle integrity
subsequent powders are used in a wide range of applications (including (Dalgleish & Corredig, 2012; de Kruif, Huppertz, Urban, & Petukhov,
infant milk formula and sports nutritional beverages and foods), their 2012; McMahon & Oommen, 2008; Horne, 2006; Wong, Camirand,
reconstitution properties can be challenging (Crowley, Kelly, Schuck, Pavlath, Parris, & Friedman, 1996).
Jeantet, & O’Mahony, 2016). Several factors contribute to their in- Chelating salts act by sequestering free calcium ions in milk-based
solubility, such as the heat treatment of the skim milk, protein and systems, and can also potentially interact with calcium associated with
mineral content, UF and DF conditions (e.g., volume concentration the CCP located within the casein micelles, to an extent dependent on
factor), spray drying temperatures, storage conditions of the subsequent the electrostatic strength and ability of the chelating salt to bind cal-
powder (e.g., time and temperature) and the rehydration process (e.g., cium. The binding of calcium through the addition of chelating salts,
temperature of reconstitution medium and mixing conditions) (Gaiani alters the distribution of calcium between the colloidal and serum
et al., 2010; Mimouni, Deeth, Whittaker, Gidley, & Bhandari, 2010; phases, which consequently influences electrostatic-mediated pro-
Richard et al., 2012). tein–protein interactions. This results in a greater net negative charge
There are a number of novel methods available for increasing the on the caseins, resulting in increased electrostatic repulsion, decreased
solubility of high protein MPC powders. Previous work (Bhaskar, Singh, protein–protein interaction and increased viscosity (De Kruif & Holt,
& Blazey, 2007) has shown that depleting calcium from MPC, using ion- 2003; Lin, Leong, Dewan, Bloomfield, & Morr, 1972; Odagiri &
exchange technology, can lead to significant increases in protein Nickerson, 1964). Depending on calcium chelating salt type and
⁎
Corresponding author.
E-mail address: [email protected] (N.A. McCarthy).
https://doi.org/10.1016/j.foodchem.2018.07.086
Received 16 January 2018; Received in revised form 10 July 2018; Accepted 12 July 2018
Available online 29 July 2018
0308-8146/ © 2018 Elsevier Ltd. All rights reserved.
O.M. Power et al. Food Chemistry 271 (2019) 136–141
concentration used, casein micelles can dissociate into single or mul- 2.4. Protein profile analysis by electrophoresis
tiple protein clusters (De Kort, Minor, Snoeren, Van Hooijdonk, & Van
Der Linden, 2009, 2011; Panouillé, Benyahia, Durand, & Nicolai, 2005; Urea-PAGE was used to separate casein proteins using the method of
Pitkowski, Nicolai, & Durand, 2008). Ornstein and Davis (1964), with a separating gel composed of 7.5%
The individual casein proteins (i.e., αs1-, αs2-, β- and κ-casein) have acrylamide, 0.375 M Tris–HCl, pH 8.8 and 4 M urea. Protein samples
different degrees of phosphorylation, with αs1-, αs2-, β- and κ-casein were prepared at a concentration of 5 mg/mL in sample buffer, and 20
having 8, 11/12, 5 and 1 phosphate residues, respectively (De Kruif & µL of sample was loaded onto the gel. After electrophoresis, the gels
Holt, 2003 Horne, 2006). A number of previous studies (Yun, Ohmiya & were stained overnight using 0.05% (w/v) Coomassie Brilliant Blue R-
Shimizu, 1982; Liu et al., 2016; McCarthy, Kelly, O'Mahony, & Fenelon, 250 in 25% (v/v) isopropanol and 10% (v/v) acetic acid. After staining,
2013) have studied the effects of dephosphorylation of casein on the gels were de-stained using a 10% (v/v) isopropanol and 10% (v/v)
charge, mineral sensitivity, protein aggregation, curd forming proper- acetic acid solution until a clear background was achieved.
ties and re-micellization of casein proteins.
The current study aimed to develop a mechanistic understanding of
2.5. Nuclear magnetic resonance
the role of phosphate groups, through dephosphorylation, on casein
proteins in MPC solutions using the advanced analytical capability of 31
Phosphate nuclear magnetic resonance (31P NMR) was conducted
phosphate nuclear magnetic resonance (31P NMR) and to control visc-
at 25 °C using a Bruker Advance III 400 NMR (Bruker UK Ltd., Coventry,
osity of MPC suspensions in which calcium ion activity had been altered
UK) located at Trinity College (Dublin, Ireland) operating at 400.2 MHz
by the addition of a calcium-chelating salt. Minimising viscosity of
for proton, 162.0 MHz for phosphorous-31 and 100.6 MHz for carbon-
concentrated casein systems, such as milk protein concentrate, is of
13 resonance. MPC powders (10–20 mg) were prepared in deuterium
academic and industrial interest in the development of protein-dense
oxide (D2O) solvent (1 mL) and NMR data was analysed using TopSpin
nutritional powders and beverages as high viscosity can negatively af-
3.5 software (Bruker UK Ltd).
fect unit operations (e.g., UF performance, spray dryer atomisation) and
finished product quality (e.g., beverage mouthfeel). Calcium chelating
salts are commonly added to concentrated casein protein solutions to 2.6. Rheological measurements of milk protein concentrate solutions
enhance powder solubilisation and heat stability; however, the con-
current swelling and viscosity increases limit their use and effectiveness Rheological measurements of MPC solutions (10%, w/w, protein)
in such applications. were carried out using a controlled-stress rheometer (AR2000ex rhe-
ometer, TA Instruments, Crawley, UK), equipped with a concentric
cylinder geometry. SHMP was prepared as described in Section 2.3 and
2. Materials and methods added to 17 mL of MPC solutions (10%, w/w, protein). Samples were
inverted ten times prior to testing to ensure a homogenous mixture.
2.1. Materials Viscosity measurements were carried out at 20 °C with pre-shearing at
100 s−1 for 10 s, followed by a peak hold step at a shear rate of 100 s−1
Milk protein concentrate (MPC) powder was obtained from a local for 2 h. Low-amplitude oscillatory shear rheological measurements of
dairy ingredient manufacturer. The protein, moisture, fat and ash the C-MPC and DP-MPC solutions containing 12.5 and 25 mM SHMP
content of the MPC were 81.4% (w/w), 4.30% (w/w), 1.40% (w/w) were also determined using a 60 mm parallel plate geometry. SHMP
and 7.80% (w/w), respectively, as provided by the manufacturer. The was added to the MPC solution to give 12.5 or 25 mM final con-
lactose content was determined by difference as 5.10% (w/w). Bovine centration and stirred for 5 min to allow adequate mixing prior to
alkaline phosphatase (BAP) enzyme was obtained from Sigma Aldrich analysis. The sample was then pre-sheared at 50 s−1 for 10 s at 20 °C to
(Vale Rd, Ballyraine Lower, Arklow, Co. Wicklow, Ireland). eliminate any shear history, followed by a time sweep at 0.25% strain
and 1 Hz frequency over 6 h, during which the rheological parameters
2.2. Reconstitution of milk protein concentrate elastic (G′) and viscous (G″) moduli were recorded.
MPC powders (250 g, 10%, w/w) were dissolved in preheated 2.7. Dynamic light scattering and particle size distribution analysis
(50 °C) distilled water aided by an overhead stirrer. Ultrasonication was
carried out using an ultrasound device (Hielscher UIP1000hd, Hielscher Particle size measurements were performed on C-MPC and DP-MPC
Ultrasonics Gmbh, Warthestraβe 21 D-14513, Berlin, Germany) using solutions with added SHMP concentrations of 0, 5, 12.5 and 25 mM
the method previously described by McCarthy, Kelly, Maher and using a Zetasizer nano (Malvern Instruments, Worcestershire, England).
Fenelon (2014) at an amplitude of 50% for 10 min to promote rehy- Measurements were conducted 1 h after SHMP addition to ensure
dration of the MPC. Sodium azide (0.02% w/w) was added to the re- equilibration. Solutions were diluted (1:50) in deionised water with a
sulting solutions to prevent microbial growth. Solutions were stirred for dispersant refractive index (RI) of 1.33 and viscosity of 0.89 mPa.s used.
4 h at 22 °C prior to storage overnight at 4 °C with low speed magnetic The refractive index used for the protein particles in the sample was
stirring. 1.45 in conjunction with an absorption value of 0.001. Experiments
were conducted in triplicate at 25 °C at a backscattering angle of 173°.
Data was displayed as the z-average (nm), correlating to the intensity-
2.3. Dephosphorylation of casein and addition of sodium
weighted mean size of all particles present in the solution.
hexametaphosphate
Reconstituted MPC solutions (10%, w/w, protein) were incubated 2.8. Statistical analysis
with BAP (1:20 w/w) at pH 6.5 and 37 °C for 3 h. A control MPC (C-
MPC) solution was prepared under the same conditions but without the Rheological measurements were analysed using a Paired T-test with
addition of the enzyme. After 3 h the reaction was terminated by a 95% confidence interval. Particle size data was statistically analysed
heating at 70 °C for 5 min to inactivate the enzyme. SHMP was dis- using one-way analysis of variance (ANOVA), with post hoc Tukey
solved in 1 mL of water as a stock solution and the pH adjusted to 6.5 analysis. The level of significance was considered as P < 0.05. All
prior to addition to the C-MPC and DP-MPC solutions to give a final statistical analysis was carried out using Minitab 17 (Minitab Inc,
concentration of 5, 12.5 or 25 mM. Coventry, United Kingdom).
137
O.M. Power et al. Food Chemistry 271 (2019) 136–141
Fig. 3. Viscosity profiles of C-MPC (black lines) and DP-MPC (grey lines) so-
lutions (10%, w/w, protein) containing 0 (—) or 5 (- - -) mM sodium hexam-
etaphosphate, measured at a shear rate of 100 s−1 at 20 °C.
Fig. 1. Urea-PAGE protein profiles of MPC (lane 1), bovine alkaline phospha-
tase enzyme (lane 2), C-MPC solution (lane 3) and DP-MPC solution (lane 4).
confirmed by previous studies conducted by Liu et al. (2016) and
McCarthy et al. (2013).
3. Results
3.1. Protein profile of non-dephosphorylated and dephosphorylated casein 3.2. Nuclear magnetic resonance spectroscopy of milk protein concentrate
solutions
Incubation of MPC with BAP for 3.5 h at 37 °C resulted in depho-
sphorylation of casein, as shown by urea-PAGE profiles in Fig. 1. Urea- Fig. 2 shows 31P NMR profiles of both C-MPC and DP-MPC solutions
PAGE separates proteins based on their charge-to-mass ratio and so with and without SHMP addition. Profiles of control MPC without
dephosphorylated protein bands were observed to have lower electro- SHMP addition (Fig. 2A) showed the calcium phosphate nano-cluster
phoretic mobility on the urea-PAGE gel (Fig. 1; lane 4) due to a re- regions (CPN) associated with the casein micelle as a broad multiplet
duction in their charge-to-mass ratio. These bands corresponded to αs1- between 1.89 and 3.37 ppm and a further broad singlet at 0.93 ppm,
and β-casein with varying degrees of dephosphorylation. Urea-PAGE attributed to non-serine bound inorganic phosphate (Pi). DP-MPC so-
profiles showed a greater reduction in the original β-casein band in- lutions (0 mM SHMP) showed a significant change in the NMR spectra
tensity, compared to αs1-casein suggesting a greater extent of depho- from that of C-MPC, with the absence of a CPN region (Fig. 2C). Fol-
sphorylation of the former casein. In the current study, BAP was chosen lowing addition of SHMP to MPC solutions, peaks were observed at
as the dephosphorylating enzyme over potato acid phosphatase (PAP) ∼−21 and −24 ppm (Fig. 2B and D). Addition of SHMP (5 mM) to the
due to a proteolytic side reaction during incubation of PAP with MPC as C-MPC solution caused a shift in the 31P spectra in the CPN region from
evident on urea-PAGE gels from preliminary trials (data not shown) and 1.76 to 3.36 ppm to 1.93–2.66 ppm, with larger and sharper peaks
31
Fig. 2. P NMR profiles of C-MPC at SHMP concentrations of 0 mM (A) and 5 mM (B) and DP-MPC at SHMP concentrations of 0 mM (C) and 5 mM (D).
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O.M. Power et al. Food Chemistry 271 (2019) 136–141
Fig. 4. Elastic, G′ (—; Pa) and viscous G″ (- - -; Pa) moduli, of C-MPC (A) and DP-MPC (B) solutions (10%, w/w, protein) containing 12.5 (■) and 25 (●) mM sodium
hexametaphosphate, measured at a frequency of 1 Hz and strain of 0.25%.
being observed also (Fig. 2B). In contrast, addition of the same level of from a shift in monomodal size distribution peaks towards larger par-
SHMP to DP-MPC created only a single large Pi peak at 0–2.0 ppm ticle size (Fig. 5B and C). However, C-MPC particle size values were
(Fig. 2D), with no evidence of CPN peaks. significantly (P < 0.05) lower at 12.5 mM SHMP addition (107.3 nm),
indicating partial dissociation of casein micelles. In contrast, the par-
3.3. Rheological measurements of milk protein concentrate solutions ticle size of DP-MPC solutions with 12.5 mM SHMP decreased to
143 nm, also indicative of micelle disintegration, albeit not to the same
Viscosity profiles of C-MPC and DP-MPC solutions without and with extent as observed in the C-MPC solution. Addition of 25 mM SHMP
added SHMP (5 mM) are shown in Fig. 3. C-MPC and DP-MPC solutions resulted in even greater micelle dissociation, as evidenced by particle
exhibited similar viscosity profiles in the absence of SHMP, with final size values for C-MPC and DP-MPC solutions of 108 and 100 nm
viscosity values of 10 and 9.0 mPa.s (P > 0.05), respectively. The ad- (P > 0.05), respectively (Fig. 5A–C).
dition of 5 mM SHMP resulted in an increase in viscosity for both C-
MPC and DP-MPC, with the viscosity of DP samples being significantly 4. Discussion
(P < 0.05) lower than those of the control sample with added SHMP
(Fig. 3). The viscosity of DP-MPC increased slowly from an initial value Previous studies (De Kruif & Holt, 2003; Lin et al., 1972; Odagiri &
of 13 mPa.s to a final viscosity of 40 mPa.s over a 2 h period, compared Nickerson, 1964) have shown the benefits of adding SHMP to con-
to the viscosity for C-MPC which increased progressively from 13 mPa.s centrated milk protein solutions prior to drying, aiding subsequent
to a final viscosity of 125 mPa.s over the same time frame (Fig. 3). powder dissolution and protein hydration. However, the benefits
Gelation properties of MPC solutions containing 12.5 and 25 mM achieved in powder dissolution can be undermined by the high visc-
SHMP are shown in Fig. 4. The C-MPC sample with 12.5 mM SHMP osity of the protein concentrate stream prior to spray drying; often re-
showed an elastic (G’) and viscous modulus (G″) crossover at 12.8 min quiring such MPC concentrates to be dried at lower solids content. The
(G′ and G″ = 5.89 Pa), with final G′ and G″ values of 557 and 219 Pa, aim of this study was to understand in more detail the fundamental
respectively, after 2.5 h (Fig. 4A). The C-MPC sample with 25 mM chemistry underlying the increased viscosity of MPC concentrates on
SHMP had a G′/G″ moduli crossover time of 18.6 min (G′ and addition of a strong polyphosphate chelating salt such as SHMP. This
G″ = 20.0 Pa), with final G′ and G″ values of 419 and 200 Pa after 2.5 h, new understanding will allow better prediction and control of viscosity
respectively. In contrast, DP-MPC with 12.5 mM SHMP formed a gel of MPC concentrate solutions.
after an extended time-frame of 20.4 min (G′ and G″ = 6.17 Pa), with SHMP is a hexadentate ligand, and hence, in milk protein systems,
final G′ and G″ moduli of 245 and 124 Pa, respectively (Fig. 4B). DP- can simultaneously chelate calcium in the aqueous phase and chelate
MPC solutions with 25 mM added SHMP formed a gel network with an calcium from CCP (De Kort, Minor, Snoeren, van Hooijdonk, & van der
elastic and viscous moduli crossover at 31.8 min (G′ and G″ = 30.5 Pa), Linden, 2011). This chelation of cations changes the ionic balance of
with final G′ and G″ values of 86.2 and 69.9 Pa, respectively (Fig. 4). the aqueous phase, thereby affecting electrostatic casein-casein inter-
actions and the structural integrity of the micelle, leading to swelling
3.4. Particle size measurements of milk protein concentrate (Fig. 5). This increase in casein micelle swelling is caused mainly by
increased electrostatic repulsion between proteins created by newly-
Data for particle size analysis of MPC solutions is shown in Fig. 5. exposed serine phosphate groups (De Kort et al., 2009, 2011; McCarthy
The z-average particle size values for C-MPC and DP-MPC solutions et al., 2017; Mizuno & Lucey, 2007; O'Connell, Grinberg, & de Kruif,
containing no SHMP were 168 and 183 nm (P > 0.05), respectively 2003; Panouille, Durand, Nicolai, Larquet, & Boisset, 2005).
(Fig. 5A), while their corresponding size distribution profiles showed NMR data showed that dephosphorylation and addition of SHMP
large monomodal peaks (Fig. 5B and C). However, C-MPC and DP-MPC significantly influenced the NMR phosphate signals in MPC solutions
solutions containing 5 mM SHMP had z-average particle size values of (Fig. 2). Boiani, McLoughlin, Auty, FitzGerald and Kelly (2017) iden-
205 and 206 nm, respectively, indicating that both samples displayed tified the effects of diafiltration water containing citrate on miceller
similar swelling behaviour upon SHMP addition (P > 0.05), as evident casein-based milk proteins with an emphasis on the phosphate signals
139
O.M. Power et al. Food Chemistry 271 (2019) 136–141
Fig. 5. Z-average particle size values for C-MPC and DP-MPC solutions (A) and particle size distribution profiles for C-MPC (B) and DP-MPC (C) solutions containing
(0, 5, 12.5 and 25 mM) sodium hexametaphosphate. Data shown is the mean value derived from triplicate analysis in each case.
using 31P NMR. The authors highlighted a peak located at ∼1.00 ppm phosphoserine peak located at 3.2 ppm to resolve into three distinctive
to be that of Pi (H2PO4− and HPO42−) along with broad, low-intensity peaks indicating that chelating action impacts on micelle integrity. This
peaks ranging from 2.0 to 3.0 ppm, identified as casein-associated can also account for the changes observed in Pi peak intensity in the
phosphate as well as a smaller peak located at ∼0.5 ppm, identified as a current study. Boiani et al. (2017) showed similar results when MPC
phosphate ester. The addition of 5 mM SHMP to C-MPC (Fig. 2B) re- manufactured using diafiltration water containing citrate was shown to
sulted in larger and sharper peaks for NMR analysis compared to C-MPC have better resolution and increased signal peaks for phosphoserine
samples without SHMP addition (Fig. 2A). This may be due to the ac- residues. When DP-MPC solutions were analysed in the absence of
tion of the SHMP chelating calcium leading to micelle swelling, and SHMP it was observed that the multiplet feature attributed to the
possible partial dissociation (Fig. 5) which would lead to an increase in phosphoserine region was absent, confirming that dephosphorylation
the exposure of serine phosphate residues, and therefore, an increase in had indeed occurred. The intensity of the Pi peak in Fig. 2C is larger and
their peak signal from NMR analysis. This experimental data demon- narrower than the peak observed in Fig. 2A, suggesting that CCP as-
strates that SHMP did impact the casein micelle structure, due to the sociated with casein proteins via phosphoserine residues had been, at
change in the distinct peak pattern of the phosphoserine residues lo- least partially, disintegrated. The addition of 5 mM SHMP to DP-MPC
cated at 2–3 ppm which resolved into a multiplet feature upon addition (Fig. 2D) showed no change at the phosphoserine site, indicating that
of 5 mM SHMP (Fig. 2B). Belton, Lyster and Richards (1985) examined no interaction occurred between SHMP and phosphoserine-bound cal-
the impact of EDTA on whole cows’ milk using 31P NMR and observed cium.
that upon EDTA addition the Pi peak increased in intensity and de- The significantly (P < 0.05) higher gel strength observed for C-
creased in width. This was likened to the chelation of free Pi in solution MPC compared to DP-MPC at 12.5 and 25 mM SHMP addition levels
by EDTA and subsequent dissociation of calcium bound by phospho- (Fig. 4A and B) could indicate that gelation brought about by SHMP
serine residues. Furthermore, the addition of EDTA caused the interactions with calcium bound to phosphoserine residues was
140
O.M. Power et al. Food Chemistry 271 (2019) 136–141
hindered in DP-MPC. Similarly, C-MPC also had higher viscosity values Development Authority (Project Number: MDDT 6714).
compared to DP-MPC solutions (Fig. 3). The lower viscosity values for
DP-MPC could be attributed to a number of possible factors. CCP has a References
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spectra data. This research was funded by Teagasc Agriculture and Food
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