Accepted Manuscript Calcium and phosphorus equilibria during acidification of skim milk at elevated temperature Glykeria Koutina, Leif H. Skibsted PII:
S0958-6946(15)00017-5
DOI:
10.1016/j.idairyj.2015.01.006
Reference:
INDA 3793
To appear in:
International Dairy Journal
Received Date: 2 December 2014 Revised Date:
8 January 2015
Accepted Date: 8 January 2015
Please cite this article as: Koutina, G., Skibsted, L.H., Calcium and phosphorus equilibria during acidification of skim milk at elevated temperature, International Dairy Journal (2015), doi: 10.1016/ j.idairyj.2015.01.006. 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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Calcium and phosphorus equilibria during acidification of skim milk at elevated temperature
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Glykeria Koutina, Leif H. Skibsted*
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Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 30, DK-
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1958 Frederiksberg C, Denmark.
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Corresponding author. Tel.: +45 3533 3221
E-mail address:
[email protected]. (L. H. Skibsted)
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Abstract
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The solubilisation of calcium and phosphorus from casein micelles was studied during skim
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milk acidification with glucono-delta-lactone. Dynamic measurements of storage modulus (G’) as a
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function of time were recorded during the formation of acid milk gels with a final pH of 4.6. An
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abnormal change in slope of the storage modulus G’ (G’shoulder) as a function of time was observed
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at 50 and 60 °C with decreasing pH. During the appearance of G’shoulder immediately after onset of
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gelation there was a large change in the concentrations of serum and micellar calcium and
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phosphorus for a change of 0.1 pH units. The onset of gelation at pH values, where the colloidal
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calcium phosphate is not fully solubilised, is coupled to casein micelle disintegration, and will
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create increased bonding between caseins forming the final acid gel with a concomitant and sudden
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solubilisation of calcium and phosphorus from the casein micelles.
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1.
Introduction
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Calcium and phosphorus are important nutrients of the mineral fraction of milk and are distributed between the serum and micellar milk phase. In the serum milk phase, calcium can be
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found as free calcium ions, Ca2+, or associated with citrate, inorganic phosphate and, to a lesser
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degree, with chloride. Phosphorus, on the other hand, is found mainly as free phosphate ions or
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associated with calcium. In the micellar phase, calcium and phosphorus are present in the form of
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colloidal calcium phosphate (CCP), which is bound to certain regions of caseins micelles and helps
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to maintain micellar integrity (Gaucheron, 2005).
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Calcium in milk, and especially Ca2+, has received considerable attention due to its major contribution to stabilisation of the casein system during milk processing and during the formation of
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acid and rennet coagulum (Fox & McSweeney, 1998; Lucey & Fox, 1993; Walstra & Jenness,
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1984). For skim milk, the molar ratio of serum to free calcium (Ca2+) is 1.71 at incubation
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temperatures between 4 and 40 °C during milk acidification (pH values between 6.6 to 4.6),
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indicating that a significant amount of liberated calcium from the casein micelles will remain as free
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ions in the serum milk phase and could be available for further interactions during the formation of
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acid coagulum (Koutina, Knudsen, Andersen, & Skibsted, 2014). In addition, the distribution of
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calcium and phosphorus between the serum and micellar milk phase can be altered by pre-heat
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treatment of milk followed by acidification or incubation of milk at specific temperatures during
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acidification and will influence the stability of casein micelles leading to structural modifications
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during dairy processing (Dalgleish & Corredig, 2012; de la Fuente, 1998; Gaucheron, 2005; Lucey
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& Horne 2009). The changes in mineral equilibrium by heat treatment can be reversed, to some
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extent, upon cooling and when enough time is allowed for re-establishment of the equilibrium
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(Holt, 1985).
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processing variables that will lead to formation of yoghurt as an important dairy product. During
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incubation of milk between 4 to 40 °C at constant pH, the amount of calcium and phosphorus in
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serum milk phase is decreased (Anema, 2009a; Dalgleish & Law, 1989; Koutina et al., 2014; Law
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& Leaver, 1998). This behaviour can be explained by the reduction in the calcium phosphate
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solubility with increasing temperature, which will lead to calcium phosphate precipitation (de la
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Fuente, Olano & Juarez, 2005; Havea, Singh, & Creamer, 2001; Singh, 2004). During milk
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acidification the net negative charge of the casein micelles decreases, weakening the electrostatic
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repulsions, and CCP is completely solubilised from the micelles at pH 5.2 and below, resulting in
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loss of integrity of casein micelles and formation of casein micelles aggregates (van Hooydonk,
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Hagedoorn, & Boerrigter, 1986b). As the pH approaches 4.6, a gel is formed by casein micelles
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aggregates linked together in a final acid coagulum network (Gastaldi, Lagaude, & de la Fuente,
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1996; Lucey, 2007). An important observation regarding milk gelation is that for milk incubated at
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temperatures lower than 40 °C, the acid gel formation starts at a pH lower than 5.1, while at
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temperatures higher than 40 °C, the acid gel formation starts at a pH higher than 5.2 (Lucey, 2007;
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Vasbinder, Rollema, Bot, & de Kruif, 2003). All types of caseins have been found to dissociate
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from the micelles at 4, 20 and 30 °C during milk acidification, with dissociation found most
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significant at 4 °C due to reduced hydrophobic interactions (Dalgleish & Law, 1988).
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The rheological properties of acid gels have been related to the strength and the number of
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bonds within the casein micelles and the spatial distribution among the micelles to form the final
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network (Anema, 2009b; Mellema, van Opheusden, & van Vliet, 2002b; Roefs & van Vliet, 1990;
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Roefs, de Groot-Mostert, & van Vliet, 1990; van Vliet, Lakemond & Visschers, 2004). During the
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formation of acid gels, dynamic measurements of gel firmness for decreasing pH have shown an
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initial lag phase prior to gelation pH, followed by a sudden increase, followed by a plateau for the
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than 40 °C, an abnormal change in slope of G’ versus time has been observed for which the term
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G’shoulder has been coined (Anema, 2009a; Dubert-Ferrandon, Niranjan, & Grandison, 2006; Lee &
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Lucey, 2004; Lucey, Tet Teo, Munro, & Singh, 1997a; Ronnegard & Dejmek, 1993).
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According to Anema (2009a) and Horne (2003), the appearance of G’shoulder is limited to
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temperatures higher than 40 °C, since gelation pH is shifted to values higher than 5.2 with CCP less
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solubilised. A role of calcium for the abnormality at higher temperature has just been documented,
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although calcium distribution may change suddenly during acidification at elevated temperatures.
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Accordingly, we have followed change in concentrations of serum calcium and phosphorus, of
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micellar calcium and phosphorus, and of free calcium before, during and after the appearance of
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G’shoulder at temperatures higher than 40 °C to investigate the importance of CCP solubilisation for
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the appearance of the G’shoulder. The overall aim of the present study is to understand the origin of
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the G’shoulder by presenting results on the solubilisation of calcium and phosphorus from the casein
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micelles in comparison with the rheological properties of skim milk during acidification at 40, 50,
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2.
Materials and methods
2.1.
Materials
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Glucono-delta-lactone (GDL), NaN3 (sodium azide), CaCl2 and NaCl of analytical grade
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were all from Sigma-Aldrich Corporation (St Louis, MO, USA). All aqueous solutions were made
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from deionised water (Mill-Q plus, Millipore Corporation, Bedford, MA, USA).
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2.2.
Acidification and ultrafiltration of incubated skim milk at 40, 50 or 60 °C
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Low heat skim milk powder (MILEX 240, Arla Foods Ingredients amba, Viby J, Denmark) was reconstituted at a level of 10% (w/w) in deionised water and after addition of sodium azide
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(0.02%), the milk was stirred overnight at 25 °C for complete hydration of casein micelles and
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equilibration of the mineral distribution. According to the manufacturers, the chemical specification
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of the product was: milk protein, 36%; lactose, 52%; milk fat, 1.25% (max.); ash, 8%; moisture,
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4%. The following day, the reconstituted skim milk was divided into 30 × 50 mL portions, and
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predetermined amounts of GDL (0.13–0.55 g 50 mL-1) to reach various pH during acidification
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were added (Skibsted & Kilde, 1971). Samples were stored, after mixing for one minute, at 40 °C
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for 5 h, at 50 °C for 4 h or at 60 °C for 3 h. Following storage, the reconstituted skim milk samples
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had final pH values from 6.4 to 4.6 as measured by a pH meter (713 pH Meter, Metrohm,
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Copenhagen, Denmark) with a glass electrode (602 Combined Metrosensor glass electrode,
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Metrohm). Ultrafiltration of the reconstituted skim milk samples (15 mL from each 50 mL portion
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of milk samples) were performed using a Beckman centrifuge (Allegra 25R, Beckman Coulter,
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Copenhagen, Denmark) and centrifuge tubes with ultra-membranes Vivaspin 20 with a molecular
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mass cut-off of 10 kDa (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), for 15 min at 6000 ×
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g at 40, 50 or 60 °C. Minerals that passed the membrane are designated herein as serum phase
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components.
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Total and serum values of calcium and phosphorus were determined in the reconstituted
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skim milk samples and in their serum phase as previously described (Koutinaet al., 2014) using IDF
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reference methods by (IDF, 2007, 2006). Micellar calcium and phosphorus were calculated by the
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assumption that the value of micellar calcium or phosphorus is equal to the total value minus the
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serum mineral value. Free calcium in serum phase of reconstituted skim samples were measured by
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an ion-selective electrode ISE25Ca with a reference REF 251 electrode (Radiometer Analytical
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SAS, Lyon, France) as previously described (Koutina et al., 2014). Data are presented as the mean
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± standard deviation (SD) of two independent replications.
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2.3.
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Gel formation and rheological properties of incubated skim milk at 40, 50 or 60 °C
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Reconstituted skim milk samples were acidified by GDL to a final pH 4.6 as recorded by a
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pH logger (pHTemp101 Data Logger, MadgeTech, Warner, NH, USA) at 40, 50 or 60 °C. During
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the pH decrease, the storage modulus (G’) at 40, 50 or 60 °C was recorded as a function of time
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using a rheometer (AR G2, TA Instrument, Elstree, UK) and methodology as previously described
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(Koutina et al., 2014). A frequency sweep test in final gels (pH 4.6) was performed at 40, 50 or 60
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°C, and the frequency values were varied from 0.1 to 5.0 Hz with a constant strain of 0.50 %. All
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measurements were within the linear viscoelastic region. Data are presented as the mean ± standard
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deviation (SD) of three independent replications.
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3.
Results and discussion
3.1.
Calcium and phosphorus equilibria during skim milk acidification
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Table 1 and 2 show the concentration of micellar, serum and free calcium, and Table 3
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shows the concentration of micellar and serum phosphorus during acidification of skim milk
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samples at 40, 50 or 60 °C. For all samples, the decrease of pH from 6.4 to 4.6 results in an increase
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in concentration of serum calcium and phosphorus (Tables 1 and 3), and of free calcium (Table 2),
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and in a decrease of micellar calcium and phosphorus (Tables 1 and 3). The decrease in pH towards
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phosphorus from the micellar to serum milk phase due to solubilisation of CCP from the casein
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micelles at pH 5.2 and below (Koutina et al., 2014; Le Graet & Gaucheron, 1999; Mekmene, Le
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Graet, & Gaucheron, 2010; van Hooydonk, Boerrigter, & Hagedoorn, 1986a; van Hooydonk et al.,
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1986b). For pH values between 5.0 and 4.6, the solubilisation of calcium from the micellar phase
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involves calcium attached to carboxyl groups of glutamate and aspartate of the caseins (Le Graet &
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Brule, 1993; van Hooydonk et al., 1986a), while the solubilisation of phosphorus from the micellar
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phase is related to the dissipation of the CCP complexes due to addition of H+ ions (Dalgleish &
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Corredig, 2012). The amount of total calcium was found to be 28.5 ± 0.9 mM and of total
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phosphorus 26.8 ± 1.5 mM, which are in accordance with the ranges normally found for milk (Fox
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& McSweeney, 1998). Notably, at 50 °C and 60 °C large changes in calcium and phosphorus
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solubilisation was observed from the micellar phase and their appearance in the serum milk phase at
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pH 5.3–5.2 and at pH 5.5–5.4, respectively, but not at 40 °C. According to Pouliot, Boulet, and
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Paquin (1989a), the amount of serum calcium will decrease with increasing temperature from 20 °C
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to 90 °C in line with the decrease in calcium phosphate solubility as temperature increases. Fig. 1a,b
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shows the plots for combinations of micellar calcium and micellar phosphorus and for combinations
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of serum calcium and free calcium including data for all temperatures and pH values.
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3.2.
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°C
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Rheological properties of incubated skim milk during and after gel formation at 40, 50 or 60
After addition of GDL, the pH of skim milk samples was found to decrease from 6.4 to 4.6
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for all temperatures investigated (Fig. 2). The higher the temperature, the shorter the time needed to
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reach pH 4.6, confirming previous studies (Lucey & Singh, 1998; Lucey, Tamehana, Singh, &
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Munro, 1998b; Won-Jae & Lucey, 2004) due to the temperature effect on the rate constant for
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hydrolysis of GDL to gluconic acid.
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The formation of acid gels from skim milk samples was followed by dynamic measurements of the evolution of storage modulus (G’) as a function of time for decreasing pH (Fig. 3). Briefly, a
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lag phase is observed during the evolution of G’ as a function of time until the gelation pH is
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reached and then a sudden and sharp increase is observed followed by a plateau (Ercili Cura et al.,
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2009; Lucey & Singh, 1998). As can be seen from Fig. 3, the G’ is low in the beginning of the
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dynamic measurements with the milk still being liquid, but when the gel starts to form and the pH
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approaches 4.6, the G’ value increases. From Fig. 3 it is seen that the final G’ values are decreasing
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with the increase in incubated temperature (Lucey, Munro & Singh, 1998a; Lucey et al., 1998b;
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McMahon, Du, McManus & Larsen 2009; Roefs et al., 1990), due to different kinetics of
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decreasing pH by GDL (Fig. 2), which will affect the building of the final gel. Additionally, the
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elevated temperatures will favour the hydrophobic interactions that have a vital role in the
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formation of acid milk gels (Lefebvre-Cases et al., 1997). The gelation pH at 40 °C was pH 5.2, at
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50 °C it was pH 5.4 and at 60 °C it was pH 5.6. In addition, an abnormal change in slope of G’
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versus time (G’shoulder) is seen for 50 °C (Fig. 3b) and 60 °C (Fig. 3c), confirming previous findings
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(Anema, 2009a; Dubert-Ferrandon et al., 2006; Horne, 2003; Lee & Lucey, 2004; Lucey et al.,
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1997a; Ronnegard & Dejmek, 1993), although no G’shoulder was observed at 40 °C. A G’shoulder was
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not detected at 40 °C, since pH for initiation of gelation was 5.2 at this lower temperature, and CCP
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is already solubilised. In contrast, at 50 °C and 60 °C, the gelation pH is higher than 5.2, and the
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CCP is only partly solubilised (Anema, 2009a; Horne, 2003).
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The development of the storage modulus, G’, for skim milk gels at pH 4.6 as a function of
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frequency for 40 °C, 50 °C and 60 °C can be seen in Fig. 4. For all elevated temperatures, the G’
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increased with an increase in frequency and the higher the temperature the lower the value of G’.
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together (intramicellar bonds) and on the spatial distribution of the casein particles (intermicellar
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bonds) to form the acid gel network (Roefs & van Vliet, 1990; Roefs et al., 1990). Hydrophobic
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interactions are accelerated by increasing temperature inside the caseins, causing the casein micelles
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to shrink (Roefs & van Vliet, 1990; Roefs et al., 1990) with smaller contact zones and weaker
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interactions among casein micelles, which will lead to low G’ values (Zoon, van Vliet & Walstra,
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1988), as were confirmed in the present study. In addition, the great solubilisation of calcium and
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phosphorus with only 0.1 pH unit of change at certain pH values at 50 °C and 60 °C (Tables 1 and
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3) is probably influencing the rearrangements and the number and strength of bonds participating to
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the gel network, forming a less firm matrix as seen from the final properties of the acid gels at these
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high temperatures.
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3.3.
Rheological properties and mineral solubilisation at G’shoulder
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A number of studies have been describing the progress of acid gelation from a kinetic point of view. Herbert, Riaublanc, Bouchet, Gallant, and Dufour (1999) suggested a 2-phase process
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requiring protein and CCP dissociation from casein micelles at pH 6.7–5.1, followed by
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reabsorption of proteins to aggregate casein micelles leading to larger aggregates at pH lower than
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5.1. Famelart, Tomazewski, Piot, and Pezennec (2004) proposed a 3-phase process requiring protein
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dissociation from casein micelles to form large and loosely protein aggregates at pH 6.7–5.4,
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followed by dissociation of CCP and formation of more compact aggregates at pH 5.3–4.9, and
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finally formation of a compact acid gel network composed of chains of compact spherical particles
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at pH lower than 4.8. McMahon et al. (2009) studied the acidification of skim milk at 10, 20, 30 and
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40 °C and suggested a 3-phase process involving dissociation of proteins in the region between pH
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6.7 and 5.4, and formation of compact colloidal particles by loosely bounds proteins for pH 5.3–4.9,
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and establishment of a final acid gel at pH lower than 4.8.
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This description of the gelation process is based on the dual-binding model proposed by Horne (1998). According to Horne’s dual-binding model, the micellar structure depends on cross
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linking of hydrophobic regions of caseins and chain growth of phosphoseryl clusters between αS1-,
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αS2- and β-caseins linking to CCP. Hydrophobic and electrostatic interactions are also essential for
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the maintenance of casein micelles structure (Horne, 1998). During lowering of the milk pH at
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temperatures lower than 30 °C, the bonds of linking CCP with caseins is broken, the electrostatic
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repulsions are decreasing, and β- and κ – caseins are solubilised from the casein micelles due to
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weaker hydrophobic interactions, which leads to gel formation (Horne, 1998, 2003).
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In the present study, the lowering of pH at temperatures higher than 40 °C clearly involves a
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different mechanism in relation to CCP solubilisation and to the amount of calcium and phosphorus
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associated with the casein micelles at the onset of acid gelation around the G’shoulder area. For heated
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milk the solubility of calcium phosphates decreases and during the heating process heat-induced
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CCP is formed, which according to Holt (1995) is associated with the existing CCP in the casein
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micelles. This heat-induced CCP may be loosely bound to the existing CCP and after the
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appearance of the gelation point, both are rapidly solubilised from the casein micelles causing the
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sudden change in the concentrations of calcium and phosphorus between the micellar and serum
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phase and the lower values of G’ for increasing temperature, confirmed in the present study (Fig. 4).
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Increasing temperature increases the strength of hydrophobic bonding causing extensive
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particle rearrangements and formation of compact aggregate clusters that will influence the
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formation of final acid gels (Bringe & Kinsella, 1987; Lucey, van Vliet, Grolle, Geurts, & Walstra,
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1997b; Lucey et al., 1998b). As part of this process a sudden liberation of calcium and phosphorus
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is observed (Tables 1 and 3), especially for 50 °C and 60 °C in the G’shoulder area. Notably, the onset
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rupture of intra micellar bonds creating different junctions in the formation of the acid gel with a
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great solubilisation of calcium and phosphorus at certain pH values. It is also worth to mention that
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even though hydrophobic interactions are important at elevated temperatures, the role of the
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decreasing charge of the proteins, due to lower pH, and the influence of the ionic interactions
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should also be take into consideration for the formation of the acid gels (Dalgleish & Law, 1989).
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A number of studies (Holt et al., 1989; Koutina et al., 2014; Mekmeneet al., 2010; van
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Hooydonk et al., 1986b) have shown a linear relationship between micellar calcium and phosphorus
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or micellar calcium and inorganic phosphate for the temperature region usually used in dairy
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technology (4–30 °C). In our study (Fig. 1 a,b) a deviation from the linear relationship is seen,
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indicated a ratio between micellar calcium and micellar phosphorus and between serum calcium and
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free calcium dependent on temperature for temperatures higher than 40 °C. This varying ratio in
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calcium and phosphorus liberation from the casein micelles should be further explored for
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technological use by the dairy industry.
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Conclusions
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4.
The appearance of the G’shoulder area at elevated temperatures during acid gelation of skim
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milk is in accordance with an increased solubilisation of calcium and phosphorus immediately after
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the onset of the acid gelation process and reveals an important role of minerals in the gelation
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process at elevated temperatures. A better understanding of the properties of the caseins in relation
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to the mineral distribution in milk is relevant for practical dairy processing and future studies should
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involve a broader range of temperatures and pH. In addition, the changes that occur in the casein
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micelles and especially in the CCP at elevated temperatures are related to aggregation and
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dissociation of the casein micelles and needs to be further investigate for possible applications in
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dairy products.
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Acknowledgement
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This work was supported by Arla Foods Amba.
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and P. F. Fox (Ed.), Advanced dairy chemistry. Vol. 3. Lactose, water, salts and minor
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gels. 2. The effect of temperature. Netherlands Milk and Dairy Journal, 42, 271-294.
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ACCEPTED MANUSCRIPT Figure legends
Fig. 1. Plots of (a) micellar phosphorus versus micellar calcium and (b) free calcium versus serum calcium for all tested temperatures (40 °C, 50 °C and 60 °C) and pH values (pH 6.4 to
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4.6).
Fig. 2. pH profiles as a function of time (min) for reconstituted skim milk during acidification
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the mean ± SD of three independent replications.
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with GDL (0.13-0.55 g 50 mL-1) at 40 °C (), 50 °C () and 60 °C (). Results are given as
Fig. 3. Storage modulus (G’) as a function of time (min) of reconstituted skim milk during acidification with GDL at (a) 40 °C, (b) 50 °C and (c) 60 °C. For 50 °C, the figure at the right shows the concentration (mM) of micellar calcium () and storage modulus, G’, () as a
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function of time for the area around the G´shoulder. For values 5.3 and 5.2 a great solubilisation of calcium from the caseins micelles is indicated, as may also be seen in Table 1. For 60 °C, the figure at the right shows the concentration (mM) of micellar calcium () and storage
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modulus, G’, () as a function of time for the area around the G´shoulder. For pH values 5.5
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and 5.4 a great solubilisation of calcium from the caseins micelles is indicated, as may also be seen in Table 1.
Fig. 4. Storage modulus (G’) as a function of frequency (Hz) of reconstituted skim milk gels at 40 °C (), 50 °C () and 60 °C (). Results are given as the mean ± SD of three independent replications.
ACCEPTED MANUSCRIPT Table 1 Concentration of micellar and serum calcium as a function of pH of reconstituted skim milk samples at 40 °C, 50 °C or 60 °C. a
Micellar calcium (mM) 40 °C
50 °C
Serum calcium (mM) 60 °C
40 °C
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50 °C
60 °C
22.90 ± 1.02 23.16 ± 0.18 22.50 ± 0.75
5.57 ± 0.51
5.31 ± 0.09
5.97 ± 0.38
5.6
17.27 ± 0.26 18.49 ± 0.39 19.99 ± 0.37
11.26 ± 0.13
9.98 ± 0.20
8.49 ± 0.18
5.5
15.81 ± 0.29 16.53 ± 1.51 18.09 ± 0.70
12.66 ± 0.15
11.95 ± 0.75 10.38 ± 0.35
5.4
14.83 ± 0.47 14.39 ± 1.51 13.43 ± 0.01
13.64 ± 0.24
14.08 ± 0.75 15.04 ± 0.01
5.3
13.26 ± 1.26 13.33 ± 0.01 12.26 ± 1.51
15.21 ± 0.63
15.15 ± 0.01 16.21 ± 0.75
5.2
11.19 ± 0.01 9.16 ± 0.94
11.30 ± 0.01
17.28 ± 0.01
19.31 ± 0.47 17.17 ± 0.01
5.1
8.61 ± 0.79
7.06 ± 0.99
9.63 ± 0.65
19.86 ± 0.39
21.41 ± 0.50 18.84 ± 0.33
5.0
5.65 ± 0.01
6.67 ± 0.56
8.59 ± 0.69
22.82 ± 0.01
21.80 ± 0.28 19.89 ± 0.34
4.9
3.16 ± 1.51
6.38 ± 0.97
8.08 ± 0.03
25.31 ± 0.75
22.09 ± 0.49 20.39 ± 0.02
4.6
2.87 ± 0.10
3.91 ± 1.56
3.38 ± 0.87
25.60 ± 0.05
24.56 ± 0.78 25.09 ± 0.43
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Values are means ± standard deviation; values are highlighted in bold font as a visual aid.
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ACCEPTED MANUSCRIPT Table 2 Concentration of free calcium as a function of pH of reconstituted skim milk samples at 40 °C, 50 °C or 60 °C. a
50 °C
60 °C
6.4
0.94 ± 0.01
0.84 ± 0.01
0.42 ± 0.01
5.6
3.88 ± 0.01
3.28 ± 0.02
1.80 ± 0.02
5.5
4.30 ± 0.02
3.87 ± 0.01
3.53 ± 0.02
5.4
4.76 ± 0.01
4.60 ± 0.02
3.87 ± 0.01
5.3
5.11 ± 0.02
5.17 ± 0.01
4.08 ± 0.02
5.2
5.68 ± 0.03
5.82 ± 0.03
4.34 ± 0.02
5.1
6.12 ± 0.03
6.76 ± 0.03
5.29 ± 0.01
5.0
6.85 ± 0.01
7.51 ± 0.01
5.42 ± 0.02
7.37 ± 0.02
8.56 ± 0.02
6.02 ± 0.01
8.04 ± 0.01
8.99 ± 0.02
7.57 ± 0.03
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4.6
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4.9
Values are means ± standard deviation.
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ACCEPTED MANUSCRIPT Table 3 Concentration of micellar and serum phosphorus as a function of pH of reconstituted skim milk samples at 40 °C, 50 °C or 60 °C. a Micellar phosphorus (mM)
Serum phosphorus (mM) 50 °C
60 °C
6.4
16.69 ± 0.25
18.32 ± 0.14 19.68 ± 0.17
7.10 ± 0.25
8.47 ± 0.14
7.11 ± 0.17
5.6
16.50 ± 0.27
17.26 ± 0.23 15.17 ± 0.72
10.29 ± 0.27
9.52 ± 0.23
11.61 ± 0.72
5.5
12.86 ± 0.09
16.47 ± 0.03 11.18 ± 0.01
13.93 ± 0.09
10.31 ± 0.03 15.61 ± 0.01
5.4
10.30 ± 0.04
16.09 ± 0.03 6.68 ± 0.34
16.48 ± 0.04
10.70 ± 0.03 20.10 ± 0.34
5.3
9.89 ± 0.14
14.96 ± 0.41 6.40 ± 0.27
16.90 ± 0.14
11.82 ± 0.41 20.39 ± 0.27
5.2
9.51 ± 0.34
9.21 ± 0.91
6.04 ± 0.03
17.28 ± 0.34
17.58 ± 0.91 20.75 ± 0.03
5.1
8.03 ± 0.13
8.57 ± 0.16
5.64 ± 0.12
18.76 ± 0.13
18.22 ± 0.16 21.14 ± 0.12
5.0
6.62 ± 0.14
7.69 ± 0.47
5.59 ± 0.04
20.17 ± 0.14
19.09 ± 0.47 21.20 ± 0.04
4.9
6.01 ± 0.35
6.58 ± 0.31
5.08 ± 0.44
20.78 ± 0.35
20.21 ± 0.31 21.70 ± 0.44
4.6
5.22 ± 0.38
5.82 ± 0.28
4.63 ± 0.16
21.56 ± 0.38
20.97 ± 0.28 22.15 ± 0.16
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40 °C
Values are means ± standard deviation; values are highlighted in bold font as a visual aid.
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60 °C
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50 °C
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Free Ca Ca (mM) (mM) Free
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Figure 2
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Time (min)
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pH:5.2
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pH:5.4
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G' (Pa)
MicellarCa Ca (mM) (mM) Micellar
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Micellar MicellarCa Ca(mM) (mM)
G' (Pa)
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The solubilization of calcium and phosphorus from casein micelles was studied during skim milk acidification with glucono-delta-lactone at 40, 50 and 60 °C. Dynamic measurements of storage modulus (G’) as a function of time were recorded during the formation of acid milk gels with a final pH of 4.6. An abnormal change in slope of G’ (G’shoulder) as a function of time was observed at 50 and 60 °C for decreasing pH. During the appearance of G’shoulder, immediately after onset of gelation, there was a large change in the concentrations of serum and micellar calcium and phosphorus for 0.1 change of pH. The onset of gelation at pH values, where the colloidal calcium phosphate is not fully solubilized, is coupled to casein micelle disintegration, and will create increased bonding between caseins forming the final acid gel with a concomitant and sudden solubilization of calcium and phosphorus from the casein micelles.