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Influence of colloidal calcium phosphate level on the microstructure and rheological properties of rennet-induced skim milk gels
LWT - Food Science and Technology xxx (2015) 1e6
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Influence of colloidal calcium phosphate level on the microstructure and rheological properties of rennet-induced skim milk gels Glykeria Koutina a, Jes C. Knudsen a, Ulf Andersen b, Leif H. Skibsted a, * a b
Food Chemistry, Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark Arla Strategic Innovation Centre, Arla Foods, DK-8220 Brabrand, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 June 2014 Received in revised form 18 February 2015 Accepted 11 March 2015 Available online xxx
Colloidal calcium phosphate is an essential part of casein micelles and being responsible for their stability. Different mineralization of casein micelles was obtained by acidification of skim milk to pH 6.5, 6.0 or 5.5, followed by a dialysis method, using simulated milk ultrafiltrate without lactose, to obtain varying levels of micellar calcium and phosphorus but constant value of pH, serum and free calcium, and serum phosphorus. Bovine chymosin was added to the skim milk samples after dialysis and microstructural and rheological properties during gel formation were recorded at 30 C. Samples after dialysis needed approximately 30 min after the addition of chymosin to form rennet gels. In addition, low micellar calcium and phosphorus values were both found to correlate with slightly less time for the gels to be formed. This information highlights the importance of CCP in the primary phase of rennet gel formation. The protein network of rennet gels after dialysis was more compact with many aggregates as demineralization decreased. The small protein particles are able to increase the potential connection points among proteins, support particle fusion and cause a compact structure. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Colloidal calcium phosphate Microscopy Rheology Rennet gel
1. Introduction Gelation of milk is a crucial step for cheese production because the milk proteins, especially caseins, are destabilized as an initial step in gel network formation. Gelation can be induced by rennet (chymosin enzyme) action, acidification or combination of acid and rennet (Lucey, Johnson, & Horne, 2003). During renneting, chymosin cuts off the N-terminal part of k-casein and para-casein micelles are formed (primary phase of rennet gel formation), while the hydrophilic parts of k-casein is released to serum milk phase mainly as caseinomacropeptide (CMP). In addition, the protein system starts to be unstable and a gel is finally formed (secondary phase of rennet coagulation) as first steps in cheesemaking (Mellema, Heesakkers, van Opheusden, & van Vliet, 2000a). The structure development of rennet gels is one of the most critical steps during cheesemaking and may lead to and control specific structural and rheological properties of the final cheese products (Zoon, van Vliet, & Walstra, 1988a). The formation of rennet gels
* Corresponding author. Tel.: þ45 3533 3221; fax: þ45 3533 3344. E-mail addresses: [email protected] (G. Koutina), [email protected] (J.C. Knudsen), [email protected] (U. Andersen), [email protected] (L.H. Skibsted).
had been an issue for many studies with special focus on rheology and microstructural properties (Ong, Dagastine, Kentish & Gras, 2012; Mellema, Walstra, van Opheusden, & van Vliet, 2000b; Zoon et al., 1988a; Renault, Gastaldi, Cuq, & de la Fuente, 2000; Frederiksen et al., 2011). Calcium is found in milk in equilibrium between the micellar and the serum milk phase. In the serum phase, calcium is present as free Ca2þ ions or associated in complexes mainly with inorganic phosphate and citrate and to lesser extent with chloride. In the micellar phase, calcium is present as a colloidal calcium phosphate (CCP) bound to the casein micelles (Gaucheron, 2005; Knudsen & Skibsted, 2010). CCP is suggested to be bound to phosphorylated serine residues of casein micelles, neutralizing their negative charge and giving them certain stability and structure (Tuinier & de Kruif, 2002; Horne, 2006; Dalgleish & Corredig, 2012). The dissociation of CCP from caseins in milk has been a main topic for numbers of studies as a way to understand the structure of casein micelles (Pyne & McGann, 1960; Ozcan-Yilsay, Horne, & Lucey, 2011; Famelart, Gauvin, Paquet, & Brule, 2009; Ozcan-Yilsay, Lee, Horne, & Lucey, 2007; Udabage, McKinnon, & Augustin, 2001). An interesting approach from Silva et al. (2013) was published describing preparation of casein suspensions with different amount of CCP while keeping pH and ionic environment in the serum milk phase constant. Silva et al. (2013) studied different physicochemical
http://dx.doi.org/10.1016/j.lwt.2015.03.035 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Koutina, G., et al., Influence of colloidal calcium phosphate level on the microstructure and rheological properties of rennet-induced skim milk gels, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.035
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G. Koutina et al. / LWT - Food Science and Technology xxx (2015) 1e6
and foaming properties of casein suspensions having different mineralization levels of primarily micellar calcium (18.5 mmol/L; 17.1 mmol/L; 15.1 mmol/L; 12.3 mmol/L and 9.5 mmol/L) and the results were addressed in relation to different amount of CCP. Demineralization of caseins was concluded to cause dissociation of casein micelles and reduced the foaming ability due to formation of small protein particles in the casein suspensions. Anema (2009) altered in similar way the amount of micellar calcium (CCP), keeping the amount of serum calcium constant, and the gelation properties of acid gels were studied. The results of that study demonstrated that the CCP was associated to the early stages of milk gelation during acidification at different temperature. Ong et al. (2011) studied the effect of increasing CCP in milk on the texture and structure of yogurt by adding NaOH. Their study showed that the yogurt texture was affected both by increasing the level of CCP and the addition of NaOH. Zoon, van Vliet, and Walstra (1988b) prepared rennet milk gels from dialyzing milk samples following the method of Pyne and McGann (1960), but the concentrations of micellar and serum calcium changed simultaneous hampering a direct relation with micellar calcium. Ozcan-Yilsay et al. (2007) add different amounts of trisodium citrate (5e40 mmol/L) to milk samples to modify both serum and micellar calcium. Then, yogurt culture was added and samples stored at 42 C until pH decreased to 4.6. They concluded that high concentration of trisodium citrate caused a weak yogurt structure with large pores. In our study, skim milk was acidified to pH 6.5, 6.0 or 5.5 followed by preparation of milk samples with different amount of micellar calcium and phosphorus while keeping the pH and the serum calcium and phosphorus constant, following dialysis method similar to the method described by Silva et al. (2013). The limited variation in pH (6.5, 6.0 and 5.5) was chosen in order to avoid micellar flocculation (Hinz, Huppertz, & Kelly, 2012) which is especially pronounced at pH around 5.2. In addition milk samples after the dialysis step were examined with respect to rheological and microstructural properties after addition of certain amount of chymosin (0.041 IMCU/ml). For rennet milk gels after dialysis, relations between the different CCP content and rheological and microstructure properties now become possible. Ultimately, understanding the changes that occur in the structure of rennet milk gels, due to micellar calcium and phosphorus, is important in order to optimize the cheese making process, and provide more insights to the properties and the structure of casein micelles. 2. Materials and methods 2.1. Chemicals Reagent-grade chemicals and distilled-deionized water (Mill-Q plus, Millipore Corporation, Bedford, MA, USA) were used throughout. 2.2. Preparation of dialyzed milk and rennet gel Pasteurized skim milk at 72 C for 15 s (0.1 g/100 ml fat) was kindly donated by Arla Foods Amba and 0.02 g/100 ml sodium azide was added to prevent bacterial growth. In addition, the skim milk was divided into smaller samples each stored at 30 C for one day. The next day the milk samples were acidified with 1.0 M HCl to pH 6.0, and 5.5 to be compare with a control sample (pH 6.5) without addition of HCl. The dilution caused by the HCl was kept constant for all samples by addition of distilled-deionized water. Normally the pH of milk is close to 6.7 at 20 C but increasing temperature to 30 C, the solubility of calcium and phosphorus decreases while decreasing CCP with a concomitant decrease in pH (Walstra &
Jenness, 1984). Control and acidified milk samples were left at 30 C for one day and then dialyzed, using a dialysis membrane with molecular weight cut-off 12e14 kDa (Spectrum Laboratories, Inc., Rancho Dominquez, Compton, CA, USA), at 4 C for 60 h against a volume of 50 times of simulated milk ultrafiltrate without addition of lactose (Jenness & Koops, 1962). The simulated milk ultrafiltrate had the following composition: 18.3 mmol/L Sodium; 39.4 mmol/L Potassium; 9.0 Calcium mmol/L; 3.2 mmol/L Magnesium; 11.6 mmol/L Phosphorus; 32.4 mmol/L Chlorium; 9.6 mmol/L Citrate; 1.0 mmol/L Sulfate and 2.2 mmol/L Carbon dioxide. The dialysis at 4 C for 60 h will not influence the rennet coagulation since hydrophobic interaction is known to be less important (Walstra, 1990). The simulated milk ultrafiltrate was changed 5 times in 60 h and then the dialyzed milk samples (milk after dialysis) were stored at 30 C for one day. After dialysis step each sample had a volume of 20 ml. Bovine chymosin (Chymax plus, 0.041 IMCU/ml, Chr. Hansen, Hørsholm, Denmark) was added at the rate of 20 mL per 5 mL at milk after dialysis and all the samples were mixed for 1 min and incubated for 60 min at 30 C to form a rennet gel. 2.3. Chemical and mineral analysis Total nitrogen content in milk samples after dialysis was determined by the Kjeldahl method (IDF, 1993). The protein content was estimated by multiplying the nitrogen content for casein by 6.38 (van Boekel & Ribadeau Dumas, 1987). pH in milk samples after dialysis was measured directly by a pH meter (713 pH Meter, Metrohm, Copenhagen, Denmark) with a glass electrode (602 Combined Metrosensor glass electrode, Metrohm). For determination of calcium and phosphorus in serum milk phase, 10 mL of milk after dialysis were used for centrifugation (Allegra 25R, Bekman Coulter, Copenhagen, Denmark) for 60 min at 2000 g at 30 C using centrifuge tubes with ultra-membranes Vivaspin 20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) with a molecular mass cut-off of 10 kDa. Total and serum contents of calcium were determined in milk after dialysis and in their serum phase using an atomic absorption spectrometric method (IDF, 2007). Micellar calcium was calculated by the following equation: Micellar calcium ¼ Total calcium e Serum calcium. Free calcium contents were determined in serum phase of milk after dialysis using an ionselective electrode ISE25Ca with a reference REF 251 electrode (Radiometer Analytical SAS, Lyon, France) at 30 C. Before use, the calcium electrode was calibrated using standards solutions of CaCl2 (0.10, 1.0, 10 and 24 mmol/L) with 80 mmol/L NaCl as background electrolyte. Determination of free calcium contents were done using the linear relationship (Nernst equation) between the electrode potential (mV) measured in the calibration solutions and the corresponding pCa value (electrode potential of calcium electrode, pCa ¼ -log [Ca2þ]) measured in the serum phase of milk after dialysis. Total and serum contents of phosphorus were determined in milk after dialysis and in their serum phase using the standard absorption spectrometry method (IDF, 2006). Micellar phosphorus was calculated by the following equation: Micellar phosphorus ¼ Total phosphorus e Serum phosphorus. 2.4. Rheological properties The rheological properties (storage modulus G0 ) of milk samples after dialysis and after addition of chymosin (Section 2.2) as a function of time (time values were varied from 0 to 60 min, with constant frequency of 0.16 Hz and strain of 0.50%) and as a function of frequency in the final rennet gels (frequency values were varied from 0.007 to 1.5 Hz with a constant strain of 0.50%) were estimated using a rheometer (AR G2, TA Instrument, Elstree, UK). In addition
Please cite this article in press as: Koutina, G., et al., Influence of colloidal calcium phosphate level on the microstructure and rheological properties of rennet-induced skim milk gels, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.035
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the parameter tan delta (the ratio of G00 to G0 ) in the final rennet gels as a function of frequency was also taken into consideration. The rheometer was equipped with temperature controlled cone-plate geometry (Cone angle 2 deg; 0 min; 0 s, Cone diameter 40 mm and Truncation 52 microM). The geometry contained 700 mL (after gently mixed with a spoon) of milk sample after dialysis and immediately after addition of chymosin. To avoid evaporation of samples a vapor trap was used. 2.5. Confocal laser scanning microscopy (CLSM) To observe the protein structure in gel after addition of chymosin in milk samples after dialysis, a protein specific stain fast green (SigmaeAldrich Corporation, St Louis, MO, USA) was used. Approximately 1.5 mL of milk samples after dialysis were mixed with fast green (4.7 mM), for 5 min and chymosin was added. Then, 1 mL was transferred to a chambered coverglass dish (Lab-TeK, Chambered Coverglass # 1.0 Borosilicate, Thermo Fisher Scientific, Rochester, NY, USA) and incubated at 30 C for 60 min. The gel samples after dialysis were then immediately inverted for CLSM analysis. The microstructures of rennet gel samples after dialysis were observed by an inverted confocal scanning laser microscopy (Leica, SP5 II, Leica Microsystems, Heidelberg, Germany). The samples were viewed using an oil immersion 63 lens and the pin-hole diameter was 1 Airy unit. The emission filter was set at 660e710 nm for fast green which was excited at a wavelength 633 nm. Micrographs of the rennet gel samples after dialysis visualize proteins in green color (due to fast green) and serum phase in black color. The micrographs were taken in size 1024 1024 pixels, having an average of four frames. Image analysis of CLSM micrographs was performed using image J software (Research Service Branch, National Institute of Health, Maryland, USA). Two micrographs were obtained for each sample and were analyzed using the “Analyze particles” step from Image J which gave a table of the green color in pixels and a final percentage of the green color, visualizing proteins. 2.6. Statistical analysis The results of all analysis were presented as the mean ± standard deviation of two independent replicates of each milk sample after dialysis and after addition of chymosin. Data were analyzed using the one-way ANOVA procedure of the SPSS statistical software (Version SPSS 19.0, 2010, IBM Danmark ApS, Kgs. Lyngby, Denmark). When a significant probability was distinguished (P < 0.05), paired comparisons between means for each parameter were carried out using Tukey's test.
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Table 1 pH values, rennet coagulation time (RCT; minutes), total, micellar, serum and free calcium (mmol/L) and total, micellar and serum phosphorus at milk samples after dialysis at 30 C. Results are present as mean ± standard deviation of two independent replicates. Means with different letters for each parameter at different encoding samples (D1, D2 and D3) differ significantly (P < 0.05). D1 pH RCT Total Calcium Micellar Calcium Serum Calcium Free Calcium Total Phosphorus Micellar Phosphorus Serum Phosphorus
6.70 29.0 24.07 15.95 8.12 1.22 26.79 15.23 11.56
D2 ± ± ± ± ± ± ± ± ±
0.01x 1x 0.12x 0.14x 0.02x 0.06x 0.18x 0.18x 0.01x
6.70 25.2 21.58 13.29 8.29 1.18 23.39 11.82 11.57
D3 ± ± ± ± ± ± ± ± ±
0.02x 0.3y 0.29y 0.53y 0.24x 0.06x 0.32y 0.26y 0.06x
6.71 23.1 18.24 10.09 8.15 1.05 21.22 9.31 11.91
± ± ± ± ± ± ± ± ±
0.01x 0.1y 0.01z 0.07z 0.07x 0.01x 0.35z 0.26z 0.10x
and micellar calcium (Table 1) and total and micellar phosphorus (Table 1) were significantly less as a function of the demineralization of casein micelles due to decrease of pH before dialysis and due to dialysis process. Fig. 1 shows the linear relationship between the micellar calcium and micellar phosphorus after dialysis with equation y ¼ 0.99x þ 1.12; R2 ¼ 0.97, with slope 0.99. 3.2. Rheological properties of rennet milk gels after dialysis Chymosin was added to milk samples after dialysis and the formation of rennet gel was recorded following the progress of G0 value as a function of time for 60 min at 30 C (Fig. 2). Fig. 2 shows the formation of rennet gel after dialysis at D1, D2 and D3, and as the micellar calcium and phosphorus decreases, the RCT is also decreasing (RCT; time directly after the addition of chymosin to milk until to the point that the baseline begins to noticeably increase in time; Bittante, 2011). In addition the RCT of rennet milk gels after dialysis was for D1, 29 ± 1x min; for D2, 25.2 ± 0.3y min, and for D3, 23.1 ± 0.1y min (Table 1). Silva et al. (2013) followed dialysis process to prepare casein suspensions with different mineralization state by using milk permeate. Silva et al. (2013) concluded that after dialysis the reduction of CCP, caused different distribution of proteins between serum and micellar phase. Especially, an increasing amount of k-casein was moved to serum phase due to CCP reduction.
3. Results and discussion 3.1. Chemical and mineral analysis of milk samples after dialysis The pH values, the RCT (rennet coagulation time) and the concentrations of total, micellar, serum and free calcium and total, micellar and serum phosphorus of control and acidified milk samples after dialysis at 30 C (as the mean ± standard deviation of two independent replicates) are presented in Table 1. The symbols D1, D2 and D3 refer to different level of demineralization of calcium and phosphorus (D1 > D2 > D3) to samples after dialysis. The protein content (gr/kg) in milk samples after dialysis was for D1, 23.5 ± 0.10x, for D2, 24.5 ± 0.04x, and for D3, 23 ± 0.05x. For samples after dialysis (D1, D2 and D3) the concentrations of serum and free calcium (Table 1) and serum phosphorus (Table 1) were constant in accordance with similar pH value, while the concentration of total
Fig. 1. Plots of micellar calcium (Ca) versus micellar phosphorus after dialysis with equation y ¼ 0.99x þ 1.12; R2 ¼ 0.97. The slope (0.99) provides information about the Micellar Ca/Micellar P molar ratio.
Please cite this article in press as: Koutina, G., et al., Influence of colloidal calcium phosphate level on the microstructure and rheological properties of rennet-induced skim milk gels, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.035
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Fig. 2. Storage modulus (G0 ) as a function of time (min) of milk samples after dialysis and after addition of chymosin at 30 C and at D1 (-), D2 (B) and D3 ( ).
Gel formation at samples after dialysis was retarded probably due to the pH after dialysis, which was for all samples close to 6.7. At this pH both casein micelles and chymosin have a negative charge (Zoon, van Vliet, & Walstra, 1989). McGann and Pyne (1960) concluded that removal of CCP from the micelles increased the viscosity of milk and caused a decrease in the rate of associations of casein components, which explains the retardation of rennet gel formation in our study (Fig. 2). Nevertheless, the reduction of CCP causes faster flocculation of casein micelles and less time to reach RCT. This can be explained by the different accessibility of k-casein by chymosin or by the different starting point of casein aggregation after the hydrolysis of k-casein. At pH 6.6 and 30 C the flocculation of casein micelles starts at least after 70% of k-casein is hydrolyzed (Dejmek & Walstra, 2004). It is also worth to mention that the primary phase of rennet gel formation was affected by the different levels of CCP demineralization and highlights the importance of CCP at the first steps of rennet gel formation. The G0 and tan delta value as a function of frequency at 30 C were estimated after 60 min from the addition of chymosin for the samples after dialysis (Figs. 3 and 4). The G0 values increased with an increase in frequency and straight lines with slopes 0.25, (D1) 0.21 (D2), and 0.28 (D3) were observed. Tan delta decreased with an increase in frequency until 0.1 Hz and then remains almost constant for higher frequency values. The higher amount of both micellar calcium and phosphorus at D1 in relation to D2 and D3, can be correlated with high values of tan delta and lower G0 values due to decrease of bonds within the casein network (Zoon et al., 1988b).
Fig. 3. Storage modulus (G0 ) as a function of frequency (Hz) of milk samples after dialysis and after addition of chymosin at 30 C and at D1 (-), D2 (B) and D3 ( ). Standard error bars are red color for D1, blue color for D2 and magenta color for D3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Silva et al. (2013) concluded that after dialysis the suspensions became more heterogenous and small protein particles were increased and casein micelles were decreased. The microstructure of the protein network of rennet gels after dialysis at 30 C (Fig. 5 A, B and C) is an “open” structure (Fig. 5A) which gradually becoming more compact (Fig. 5B) with many protein aggregates (Fig. 5C). Low values of calcium and phosphorus in the micelles may cause a compact rennet gel structure (Fig 5C). Silva et al. (2013) found after dialysis an increasing number of casein micelles (between 20 and 50 nm) during demineralization and especially at D3. In our study the small protein particles can increase the potential connection points among proteins, promote particle fusion and cause a compact rennet gel in accordance with results presented by Mellema et al. (2000a). In addition even though the G0 values was almost similar for all rennet gels after dialysis (Fig. 3) the
3.3. Microstructure properties of rennet milk gels after dialysis The microstructure properties of rennet milk gels after dialysis were observed by confocal laser scanning microscopy at 30 C. Micrographs of the rennet gels after dialysis visualize proteins in green color and serum phase in black color (Fig. 5). Fig. 5A (D1), 5B (D2) and 5C (D3) visualize the protein structure of rennet gels after dialysis and as the micellar calcium and phosphorus decreases the protein network is becoming more compact and dense with many protein aggregates (especially for micrograph C), but still with an open structure as can be observed for micrographs A and B. After analysis of particles by Image J the percentage of green color (visualize the proteins) of each micrograph is 44.4% ± 1.4x (A, D1); 49.4% ± 1.4x (B, D2), and 75.3% ± 3.2y (C, D3).
Fig. 4. tan delta as a function of frequency (Hz) of milk samples after dialysis and after addition of chymosin at 30 C and at D1 (-), D2 (B) and D3 ( ). Standard error bars are red color for D1, blue color for D2 and magenta color for D3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Koutina, G., et al., Influence of colloidal calcium phosphate level on the microstructure and rheological properties of rennet-induced skim milk gels, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.035
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Fig. 5. CLSM micrographs showing the microstructure of gel samples at 30 C after dialysis (A, D1; B, D2; C, D3). The fast green stained protein appears green in all micrographs. Information inside the parenthesis indicate the degree of demineralization of each sample encode with certain letters and the analyze particles step from Image J as mean (%) ± standard deviation. Means with different letters in each column differ significantly (P < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
micrographs from microscopy (Fig. 5 A, B and C) show a more pronounce effect of decreasing the amount of CCP in the structure of rennet gel. This indicates that the bonds represented by G0 value is the same for different amount of CCP but the distribution of proteins forming the gel network having an increasing density for decreasing CCP levels, causing different visualization in the microscope.
4. Conclusions In order to understand the role of CCP during the formation of rennet gel, information about the casein micelles and their properties was obtained during the formation of gel at 30 C after a specific dialysis process. Even though the amount of micellar calcium and phosphorus was modified, the rennet gels needed almost 30 min to be formed, arising questions regarding the importance of micellar phase in relation to the serum milk phase, which in this study was kept constant. Still this study documented for first time that the CCP demineralization affected the primary phase of rennet gel formation. Nevertheless the decreasing amount of micellar calcium and phosphorus cause a more compact and dense structure with many proteins aggregates. Further studies will be focused on more detail investigation during ageing of rennet gel prior to formation of cheese structure and also include variations in the protein concentration.
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Ozcan-Yilsay, T., Horne, D., & Lucey, J. A. (2011). Effect of increasing the colloidal calcium phosphate of milk on the texture and microstructure of yogurt. Journal of Dairy Science, 94, 5278e5288. Ozcan-Yilsay, T., Lee, W. J., Horne, D., & Lucey, J. A. (2007). Effect of trisodium citrate on rheological and physical properties and microstructure of yogurt. Journal of Dairy Science, 90, 1644e1652. Pyne, G. T., & McGann, T. C. A. (1960). The colloidal phosphate of milk. 2. Influence of citrate. Journal of Dairy Research, 27, 9e17. Renault, C., Gastaldi, E., Cuq, J. L., & de la Fuente, B. T. (2000). Effect of temperature of milk acidification on rennet gel properties. Journal of Food Science, 65, 630e634. Silva, N. N., Piot, M., de Carvalho, A. F., Violleau, F., Fameau, A. L., & Gaucheron, F. (2013). pH-induced demineralization of casein micelles modifies their physicochemical and foaming properties. Food Hydrocolloids, 32, 322e330. Tuinier, R., & de Kruif, C. G. (2002). Stability of casein micelles in milk. Journal of Chemical Physics, 117, 1290e1295. Udabage, P., McKinnon, I. R., & Augustin, M. A. (2001). Effects of mineral salts and calcium chelating agents on the gelation of renneted skim milk. Journal of Dairy Science, 84, 1569e1575. Walstra, P. (1990). On the stability of casein micelles. Journal of Dairy Science, 73, 1965e1979. Walstra, P., & Jenness, R. (1984). Dairy chemistry and physics (pp. 163e192). New York, USA: John Wiley & Sons. Zoon, P., van Vliet, T., & Walstra, P. (1988a). Rheological properties of rennetinduced skim milk gels .1. Introduction. Netherlands Milk and Dairy Journal, 42, 249e269. Zoon, P., van Vliet, T., & Walstra, P. (1988b). Rheological properties of rennetinduced skim milk gels .3. The effect of calcium and phosphate. Netherlands Milk and Dairy Journal, 42, 295e312. Zoon, P., van Vliet, T., & Walstra, P. (1989). Rheological properties of rennet-induced skim milk gels .4. The effect of pH and NaCl. Netherlands Milk and Dairy Journal, 43, 17e34.
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Influence of colloidal calcium phosphate level on the microstructure and rheological properties of rennet-induced skim milk gels Glykeria Koutina a, Jes C. Knudsen a, Ulf Andersen b, Leif H. Skibsted a, * a b
Food Chemistry, Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark Arla Strategic Innovation Centre, Arla Foods, DK-8220 Brabrand, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 June 2014 Received in revised form 18 February 2015 Accepted 11 March 2015 Available online xxx
Colloidal calcium phosphate is an essential part of casein micelles and being responsible for their stability. Different mineralization of casein micelles was obtained by acidification of skim milk to pH 6.5, 6.0 or 5.5, followed by a dialysis method, using simulated milk ultrafiltrate without lactose, to obtain varying levels of micellar calcium and phosphorus but constant value of pH, serum and free calcium, and serum phosphorus. Bovine chymosin was added to the skim milk samples after dialysis and microstructural and rheological properties during gel formation were recorded at 30 C. Samples after dialysis needed approximately 30 min after the addition of chymosin to form rennet gels. In addition, low micellar calcium and phosphorus values were both found to correlate with slightly less time for the gels to be formed. This information highlights the importance of CCP in the primary phase of rennet gel formation. The protein network of rennet gels after dialysis was more compact with many aggregates as demineralization decreased. The small protein particles are able to increase the potential connection points among proteins, support particle fusion and cause a compact structure. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Colloidal calcium phosphate Microscopy Rheology Rennet gel
1. Introduction Gelation of milk is a crucial step for cheese production because the milk proteins, especially caseins, are destabilized as an initial step in gel network formation. Gelation can be induced by rennet (chymosin enzyme) action, acidification or combination of acid and rennet (Lucey, Johnson, & Horne, 2003). During renneting, chymosin cuts off the N-terminal part of k-casein and para-casein micelles are formed (primary phase of rennet gel formation), while the hydrophilic parts of k-casein is released to serum milk phase mainly as caseinomacropeptide (CMP). In addition, the protein system starts to be unstable and a gel is finally formed (secondary phase of rennet coagulation) as first steps in cheesemaking (Mellema, Heesakkers, van Opheusden, & van Vliet, 2000a). The structure development of rennet gels is one of the most critical steps during cheesemaking and may lead to and control specific structural and rheological properties of the final cheese products (Zoon, van Vliet, & Walstra, 1988a). The formation of rennet gels
* Corresponding author. Tel.: þ45 3533 3221; fax: þ45 3533 3344. E-mail addresses: [email protected] (G. Koutina), [email protected] (J.C. Knudsen), [email protected] (U. Andersen), [email protected] (L.H. Skibsted).
had been an issue for many studies with special focus on rheology and microstructural properties (Ong, Dagastine, Kentish & Gras, 2012; Mellema, Walstra, van Opheusden, & van Vliet, 2000b; Zoon et al., 1988a; Renault, Gastaldi, Cuq, & de la Fuente, 2000; Frederiksen et al., 2011). Calcium is found in milk in equilibrium between the micellar and the serum milk phase. In the serum phase, calcium is present as free Ca2þ ions or associated in complexes mainly with inorganic phosphate and citrate and to lesser extent with chloride. In the micellar phase, calcium is present as a colloidal calcium phosphate (CCP) bound to the casein micelles (Gaucheron, 2005; Knudsen & Skibsted, 2010). CCP is suggested to be bound to phosphorylated serine residues of casein micelles, neutralizing their negative charge and giving them certain stability and structure (Tuinier & de Kruif, 2002; Horne, 2006; Dalgleish & Corredig, 2012). The dissociation of CCP from caseins in milk has been a main topic for numbers of studies as a way to understand the structure of casein micelles (Pyne & McGann, 1960; Ozcan-Yilsay, Horne, & Lucey, 2011; Famelart, Gauvin, Paquet, & Brule, 2009; Ozcan-Yilsay, Lee, Horne, & Lucey, 2007; Udabage, McKinnon, & Augustin, 2001). An interesting approach from Silva et al. (2013) was published describing preparation of casein suspensions with different amount of CCP while keeping pH and ionic environment in the serum milk phase constant. Silva et al. (2013) studied different physicochemical
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Please cite this article in press as: Koutina, G., et al., Influence of colloidal calcium phosphate level on the microstructure and rheological properties of rennet-induced skim milk gels, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.035
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and foaming properties of casein suspensions having different mineralization levels of primarily micellar calcium (18.5 mmol/L; 17.1 mmol/L; 15.1 mmol/L; 12.3 mmol/L and 9.5 mmol/L) and the results were addressed in relation to different amount of CCP. Demineralization of caseins was concluded to cause dissociation of casein micelles and reduced the foaming ability due to formation of small protein particles in the casein suspensions. Anema (2009) altered in similar way the amount of micellar calcium (CCP), keeping the amount of serum calcium constant, and the gelation properties of acid gels were studied. The results of that study demonstrated that the CCP was associated to the early stages of milk gelation during acidification at different temperature. Ong et al. (2011) studied the effect of increasing CCP in milk on the texture and structure of yogurt by adding NaOH. Their study showed that the yogurt texture was affected both by increasing the level of CCP and the addition of NaOH. Zoon, van Vliet, and Walstra (1988b) prepared rennet milk gels from dialyzing milk samples following the method of Pyne and McGann (1960), but the concentrations of micellar and serum calcium changed simultaneous hampering a direct relation with micellar calcium. Ozcan-Yilsay et al. (2007) add different amounts of trisodium citrate (5e40 mmol/L) to milk samples to modify both serum and micellar calcium. Then, yogurt culture was added and samples stored at 42 C until pH decreased to 4.6. They concluded that high concentration of trisodium citrate caused a weak yogurt structure with large pores. In our study, skim milk was acidified to pH 6.5, 6.0 or 5.5 followed by preparation of milk samples with different amount of micellar calcium and phosphorus while keeping the pH and the serum calcium and phosphorus constant, following dialysis method similar to the method described by Silva et al. (2013). The limited variation in pH (6.5, 6.0 and 5.5) was chosen in order to avoid micellar flocculation (Hinz, Huppertz, & Kelly, 2012) which is especially pronounced at pH around 5.2. In addition milk samples after the dialysis step were examined with respect to rheological and microstructural properties after addition of certain amount of chymosin (0.041 IMCU/ml). For rennet milk gels after dialysis, relations between the different CCP content and rheological and microstructure properties now become possible. Ultimately, understanding the changes that occur in the structure of rennet milk gels, due to micellar calcium and phosphorus, is important in order to optimize the cheese making process, and provide more insights to the properties and the structure of casein micelles. 2. Materials and methods 2.1. Chemicals Reagent-grade chemicals and distilled-deionized water (Mill-Q plus, Millipore Corporation, Bedford, MA, USA) were used throughout. 2.2. Preparation of dialyzed milk and rennet gel Pasteurized skim milk at 72 C for 15 s (0.1 g/100 ml fat) was kindly donated by Arla Foods Amba and 0.02 g/100 ml sodium azide was added to prevent bacterial growth. In addition, the skim milk was divided into smaller samples each stored at 30 C for one day. The next day the milk samples were acidified with 1.0 M HCl to pH 6.0, and 5.5 to be compare with a control sample (pH 6.5) without addition of HCl. The dilution caused by the HCl was kept constant for all samples by addition of distilled-deionized water. Normally the pH of milk is close to 6.7 at 20 C but increasing temperature to 30 C, the solubility of calcium and phosphorus decreases while decreasing CCP with a concomitant decrease in pH (Walstra &
Jenness, 1984). Control and acidified milk samples were left at 30 C for one day and then dialyzed, using a dialysis membrane with molecular weight cut-off 12e14 kDa (Spectrum Laboratories, Inc., Rancho Dominquez, Compton, CA, USA), at 4 C for 60 h against a volume of 50 times of simulated milk ultrafiltrate without addition of lactose (Jenness & Koops, 1962). The simulated milk ultrafiltrate had the following composition: 18.3 mmol/L Sodium; 39.4 mmol/L Potassium; 9.0 Calcium mmol/L; 3.2 mmol/L Magnesium; 11.6 mmol/L Phosphorus; 32.4 mmol/L Chlorium; 9.6 mmol/L Citrate; 1.0 mmol/L Sulfate and 2.2 mmol/L Carbon dioxide. The dialysis at 4 C for 60 h will not influence the rennet coagulation since hydrophobic interaction is known to be less important (Walstra, 1990). The simulated milk ultrafiltrate was changed 5 times in 60 h and then the dialyzed milk samples (milk after dialysis) were stored at 30 C for one day. After dialysis step each sample had a volume of 20 ml. Bovine chymosin (Chymax plus, 0.041 IMCU/ml, Chr. Hansen, Hørsholm, Denmark) was added at the rate of 20 mL per 5 mL at milk after dialysis and all the samples were mixed for 1 min and incubated for 60 min at 30 C to form a rennet gel. 2.3. Chemical and mineral analysis Total nitrogen content in milk samples after dialysis was determined by the Kjeldahl method (IDF, 1993). The protein content was estimated by multiplying the nitrogen content for casein by 6.38 (van Boekel & Ribadeau Dumas, 1987). pH in milk samples after dialysis was measured directly by a pH meter (713 pH Meter, Metrohm, Copenhagen, Denmark) with a glass electrode (602 Combined Metrosensor glass electrode, Metrohm). For determination of calcium and phosphorus in serum milk phase, 10 mL of milk after dialysis were used for centrifugation (Allegra 25R, Bekman Coulter, Copenhagen, Denmark) for 60 min at 2000 g at 30 C using centrifuge tubes with ultra-membranes Vivaspin 20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) with a molecular mass cut-off of 10 kDa. Total and serum contents of calcium were determined in milk after dialysis and in their serum phase using an atomic absorption spectrometric method (IDF, 2007). Micellar calcium was calculated by the following equation: Micellar calcium ¼ Total calcium e Serum calcium. Free calcium contents were determined in serum phase of milk after dialysis using an ionselective electrode ISE25Ca with a reference REF 251 electrode (Radiometer Analytical SAS, Lyon, France) at 30 C. Before use, the calcium electrode was calibrated using standards solutions of CaCl2 (0.10, 1.0, 10 and 24 mmol/L) with 80 mmol/L NaCl as background electrolyte. Determination of free calcium contents were done using the linear relationship (Nernst equation) between the electrode potential (mV) measured in the calibration solutions and the corresponding pCa value (electrode potential of calcium electrode, pCa ¼ -log [Ca2þ]) measured in the serum phase of milk after dialysis. Total and serum contents of phosphorus were determined in milk after dialysis and in their serum phase using the standard absorption spectrometry method (IDF, 2006). Micellar phosphorus was calculated by the following equation: Micellar phosphorus ¼ Total phosphorus e Serum phosphorus. 2.4. Rheological properties The rheological properties (storage modulus G0 ) of milk samples after dialysis and after addition of chymosin (Section 2.2) as a function of time (time values were varied from 0 to 60 min, with constant frequency of 0.16 Hz and strain of 0.50%) and as a function of frequency in the final rennet gels (frequency values were varied from 0.007 to 1.5 Hz with a constant strain of 0.50%) were estimated using a rheometer (AR G2, TA Instrument, Elstree, UK). In addition
Please cite this article in press as: Koutina, G., et al., Influence of colloidal calcium phosphate level on the microstructure and rheological properties of rennet-induced skim milk gels, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.035
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the parameter tan delta (the ratio of G00 to G0 ) in the final rennet gels as a function of frequency was also taken into consideration. The rheometer was equipped with temperature controlled cone-plate geometry (Cone angle 2 deg; 0 min; 0 s, Cone diameter 40 mm and Truncation 52 microM). The geometry contained 700 mL (after gently mixed with a spoon) of milk sample after dialysis and immediately after addition of chymosin. To avoid evaporation of samples a vapor trap was used. 2.5. Confocal laser scanning microscopy (CLSM) To observe the protein structure in gel after addition of chymosin in milk samples after dialysis, a protein specific stain fast green (SigmaeAldrich Corporation, St Louis, MO, USA) was used. Approximately 1.5 mL of milk samples after dialysis were mixed with fast green (4.7 mM), for 5 min and chymosin was added. Then, 1 mL was transferred to a chambered coverglass dish (Lab-TeK, Chambered Coverglass # 1.0 Borosilicate, Thermo Fisher Scientific, Rochester, NY, USA) and incubated at 30 C for 60 min. The gel samples after dialysis were then immediately inverted for CLSM analysis. The microstructures of rennet gel samples after dialysis were observed by an inverted confocal scanning laser microscopy (Leica, SP5 II, Leica Microsystems, Heidelberg, Germany). The samples were viewed using an oil immersion 63 lens and the pin-hole diameter was 1 Airy unit. The emission filter was set at 660e710 nm for fast green which was excited at a wavelength 633 nm. Micrographs of the rennet gel samples after dialysis visualize proteins in green color (due to fast green) and serum phase in black color. The micrographs were taken in size 1024 1024 pixels, having an average of four frames. Image analysis of CLSM micrographs was performed using image J software (Research Service Branch, National Institute of Health, Maryland, USA). Two micrographs were obtained for each sample and were analyzed using the “Analyze particles” step from Image J which gave a table of the green color in pixels and a final percentage of the green color, visualizing proteins. 2.6. Statistical analysis The results of all analysis were presented as the mean ± standard deviation of two independent replicates of each milk sample after dialysis and after addition of chymosin. Data were analyzed using the one-way ANOVA procedure of the SPSS statistical software (Version SPSS 19.0, 2010, IBM Danmark ApS, Kgs. Lyngby, Denmark). When a significant probability was distinguished (P < 0.05), paired comparisons between means for each parameter were carried out using Tukey's test.
3
Table 1 pH values, rennet coagulation time (RCT; minutes), total, micellar, serum and free calcium (mmol/L) and total, micellar and serum phosphorus at milk samples after dialysis at 30 C. Results are present as mean ± standard deviation of two independent replicates. Means with different letters for each parameter at different encoding samples (D1, D2 and D3) differ significantly (P < 0.05). D1 pH RCT Total Calcium Micellar Calcium Serum Calcium Free Calcium Total Phosphorus Micellar Phosphorus Serum Phosphorus
6.70 29.0 24.07 15.95 8.12 1.22 26.79 15.23 11.56
D2 ± ± ± ± ± ± ± ± ±
0.01x 1x 0.12x 0.14x 0.02x 0.06x 0.18x 0.18x 0.01x
6.70 25.2 21.58 13.29 8.29 1.18 23.39 11.82 11.57
D3 ± ± ± ± ± ± ± ± ±
0.02x 0.3y 0.29y 0.53y 0.24x 0.06x 0.32y 0.26y 0.06x
6.71 23.1 18.24 10.09 8.15 1.05 21.22 9.31 11.91
± ± ± ± ± ± ± ± ±
0.01x 0.1y 0.01z 0.07z 0.07x 0.01x 0.35z 0.26z 0.10x
and micellar calcium (Table 1) and total and micellar phosphorus (Table 1) were significantly less as a function of the demineralization of casein micelles due to decrease of pH before dialysis and due to dialysis process. Fig. 1 shows the linear relationship between the micellar calcium and micellar phosphorus after dialysis with equation y ¼ 0.99x þ 1.12; R2 ¼ 0.97, with slope 0.99. 3.2. Rheological properties of rennet milk gels after dialysis Chymosin was added to milk samples after dialysis and the formation of rennet gel was recorded following the progress of G0 value as a function of time for 60 min at 30 C (Fig. 2). Fig. 2 shows the formation of rennet gel after dialysis at D1, D2 and D3, and as the micellar calcium and phosphorus decreases, the RCT is also decreasing (RCT; time directly after the addition of chymosin to milk until to the point that the baseline begins to noticeably increase in time; Bittante, 2011). In addition the RCT of rennet milk gels after dialysis was for D1, 29 ± 1x min; for D2, 25.2 ± 0.3y min, and for D3, 23.1 ± 0.1y min (Table 1). Silva et al. (2013) followed dialysis process to prepare casein suspensions with different mineralization state by using milk permeate. Silva et al. (2013) concluded that after dialysis the reduction of CCP, caused different distribution of proteins between serum and micellar phase. Especially, an increasing amount of k-casein was moved to serum phase due to CCP reduction.
3. Results and discussion 3.1. Chemical and mineral analysis of milk samples after dialysis The pH values, the RCT (rennet coagulation time) and the concentrations of total, micellar, serum and free calcium and total, micellar and serum phosphorus of control and acidified milk samples after dialysis at 30 C (as the mean ± standard deviation of two independent replicates) are presented in Table 1. The symbols D1, D2 and D3 refer to different level of demineralization of calcium and phosphorus (D1 > D2 > D3) to samples after dialysis. The protein content (gr/kg) in milk samples after dialysis was for D1, 23.5 ± 0.10x, for D2, 24.5 ± 0.04x, and for D3, 23 ± 0.05x. For samples after dialysis (D1, D2 and D3) the concentrations of serum and free calcium (Table 1) and serum phosphorus (Table 1) were constant in accordance with similar pH value, while the concentration of total
Fig. 1. Plots of micellar calcium (Ca) versus micellar phosphorus after dialysis with equation y ¼ 0.99x þ 1.12; R2 ¼ 0.97. The slope (0.99) provides information about the Micellar Ca/Micellar P molar ratio.
Please cite this article in press as: Koutina, G., et al., Influence of colloidal calcium phosphate level on the microstructure and rheological properties of rennet-induced skim milk gels, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.035
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Fig. 2. Storage modulus (G0 ) as a function of time (min) of milk samples after dialysis and after addition of chymosin at 30 C and at D1 (-), D2 (B) and D3 ( ).
Gel formation at samples after dialysis was retarded probably due to the pH after dialysis, which was for all samples close to 6.7. At this pH both casein micelles and chymosin have a negative charge (Zoon, van Vliet, & Walstra, 1989). McGann and Pyne (1960) concluded that removal of CCP from the micelles increased the viscosity of milk and caused a decrease in the rate of associations of casein components, which explains the retardation of rennet gel formation in our study (Fig. 2). Nevertheless, the reduction of CCP causes faster flocculation of casein micelles and less time to reach RCT. This can be explained by the different accessibility of k-casein by chymosin or by the different starting point of casein aggregation after the hydrolysis of k-casein. At pH 6.6 and 30 C the flocculation of casein micelles starts at least after 70% of k-casein is hydrolyzed (Dejmek & Walstra, 2004). It is also worth to mention that the primary phase of rennet gel formation was affected by the different levels of CCP demineralization and highlights the importance of CCP at the first steps of rennet gel formation. The G0 and tan delta value as a function of frequency at 30 C were estimated after 60 min from the addition of chymosin for the samples after dialysis (Figs. 3 and 4). The G0 values increased with an increase in frequency and straight lines with slopes 0.25, (D1) 0.21 (D2), and 0.28 (D3) were observed. Tan delta decreased with an increase in frequency until 0.1 Hz and then remains almost constant for higher frequency values. The higher amount of both micellar calcium and phosphorus at D1 in relation to D2 and D3, can be correlated with high values of tan delta and lower G0 values due to decrease of bonds within the casein network (Zoon et al., 1988b).
Fig. 3. Storage modulus (G0 ) as a function of frequency (Hz) of milk samples after dialysis and after addition of chymosin at 30 C and at D1 (-), D2 (B) and D3 ( ). Standard error bars are red color for D1, blue color for D2 and magenta color for D3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Silva et al. (2013) concluded that after dialysis the suspensions became more heterogenous and small protein particles were increased and casein micelles were decreased. The microstructure of the protein network of rennet gels after dialysis at 30 C (Fig. 5 A, B and C) is an “open” structure (Fig. 5A) which gradually becoming more compact (Fig. 5B) with many protein aggregates (Fig. 5C). Low values of calcium and phosphorus in the micelles may cause a compact rennet gel structure (Fig 5C). Silva et al. (2013) found after dialysis an increasing number of casein micelles (between 20 and 50 nm) during demineralization and especially at D3. In our study the small protein particles can increase the potential connection points among proteins, promote particle fusion and cause a compact rennet gel in accordance with results presented by Mellema et al. (2000a). In addition even though the G0 values was almost similar for all rennet gels after dialysis (Fig. 3) the
3.3. Microstructure properties of rennet milk gels after dialysis The microstructure properties of rennet milk gels after dialysis were observed by confocal laser scanning microscopy at 30 C. Micrographs of the rennet gels after dialysis visualize proteins in green color and serum phase in black color (Fig. 5). Fig. 5A (D1), 5B (D2) and 5C (D3) visualize the protein structure of rennet gels after dialysis and as the micellar calcium and phosphorus decreases the protein network is becoming more compact and dense with many protein aggregates (especially for micrograph C), but still with an open structure as can be observed for micrographs A and B. After analysis of particles by Image J the percentage of green color (visualize the proteins) of each micrograph is 44.4% ± 1.4x (A, D1); 49.4% ± 1.4x (B, D2), and 75.3% ± 3.2y (C, D3).
Fig. 4. tan delta as a function of frequency (Hz) of milk samples after dialysis and after addition of chymosin at 30 C and at D1 (-), D2 (B) and D3 ( ). Standard error bars are red color for D1, blue color for D2 and magenta color for D3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Koutina, G., et al., Influence of colloidal calcium phosphate level on the microstructure and rheological properties of rennet-induced skim milk gels, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.035
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Fig. 5. CLSM micrographs showing the microstructure of gel samples at 30 C after dialysis (A, D1; B, D2; C, D3). The fast green stained protein appears green in all micrographs. Information inside the parenthesis indicate the degree of demineralization of each sample encode with certain letters and the analyze particles step from Image J as mean (%) ± standard deviation. Means with different letters in each column differ significantly (P < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
micrographs from microscopy (Fig. 5 A, B and C) show a more pronounce effect of decreasing the amount of CCP in the structure of rennet gel. This indicates that the bonds represented by G0 value is the same for different amount of CCP but the distribution of proteins forming the gel network having an increasing density for decreasing CCP levels, causing different visualization in the microscope.
4. Conclusions In order to understand the role of CCP during the formation of rennet gel, information about the casein micelles and their properties was obtained during the formation of gel at 30 C after a specific dialysis process. Even though the amount of micellar calcium and phosphorus was modified, the rennet gels needed almost 30 min to be formed, arising questions regarding the importance of micellar phase in relation to the serum milk phase, which in this study was kept constant. Still this study documented for first time that the CCP demineralization affected the primary phase of rennet gel formation. Nevertheless the decreasing amount of micellar calcium and phosphorus cause a more compact and dense structure with many proteins aggregates. Further studies will be focused on more detail investigation during ageing of rennet gel prior to formation of cheese structure and also include variations in the protein concentration.
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Please cite this article in press as: Koutina, G., et al., Influence of colloidal calcium phosphate level on the microstructure and rheological properties of rennet-induced skim milk gels, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.035