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Change in molecular structure and dynamics of protein in milk protein concentrate powder upon ageing by solid-state carbon NMR
Food Hydrocolloids 44 (2015) 66e70
Contents lists available at ScienceDirect
Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Change in molecular structure and dynamics of protein in milk protein concentrate powder upon ageing by solid-state carbon NMR Enamul Haque a, *, Bhesh R. Bhandari b, Michael J. Gidley c, Hilton C. Deeth b, Andrew K. Whittaker d a
School of Applied Sciences, RMIT University, VIC 3000, Australia School of Agriculture and Food Sciences, University of Queensland, QLD 4072 Australia Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, University of Queensland, QLD 4072, Australia d Centre for Advanced Imaging and Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland, QLD 4072, Australia b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 May 2014 Accepted 9 September 2014 Available online 18 September 2014
Instability of proteins in dry form causes solubility loss of milk protein concentrate (MPC) powder upon ageing. High resolution solid state NMR techniques were used to investigate the changes in molecular structure and dynamics of proteins in MPC with varying moisture content (5.5e16.5% w/w) and storage period. The results indicate a slight higher rigidity of molecular domains of protein molecules of nonaged MPC compared to that of the long aged (at 25 C) MPC. It could be suggested from this observation that long-term storage at high relative humidity (RH) may reduce rigidity of the molecular domains due to interaction with water rather than short-term storage at high RH. This may indicate increased molecular mobility of backbone and side chains of protein molecules due to plasticization during ageing which could facilitate proteineprotein interaction and protein denaturation. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Solid-state carbon NMR Milk protein concentrate Water activity Molecular mobility Solubility
1. Introduction Milk protein concentrate (MPC) is a dairy-derived powder with high protein content, up to 85%. MPC powders are used as a food ingredient in a variety of applications for their high nutritional value and favourable functional properties. Generally, MPC powders exhibit a gradual loss of solubility upon storage (Anema, Pinder, Hunter, & Hemar, 2006; Haque et al., 2010; Havea, 2006; Mimouni, Deeth, Whittaker, Gidley, & Bhandari, 2009). Changes in the structure and dynamics of protein molecules/segments in MPC powder could occur upon storage and this could have some link with solubility loss of MPC upon ageing. Solid-state carbon NMR techniques can be used to study these changes in the structure and dynamic features of protein molecules in MPC powder after storage at different conditions. This will provide an insight into the underlying molecular mechanism of protein aggregation that is most likely responsible for the loss of solubility on storage of MPC. Solid-state NMR has widespread applications in various fields including dairy and other food products (Baldus, 2007; Belloque &
* Corresponding author. E-mail address: [email protected] (E. Haque). http://dx.doi.org/10.1016/j.foodhyd.2014.09.022 0268-005X/© 2014 Elsevier Ltd. All rights reserved.
Ramos, 1999; Kakalis, Kumosinski, & Farrell, 1994). High resolution solid-state carbon NMR is a useful tool that can be used to monitor the mobility (or rigidity) of molecular segments in the backbone as well as side chains of protein molecules. This technique has been used to probe the calcium binding sites of casein and to ascertain the dynamic state of amino acid residues within casein micelles (Kakalis, Kumosinski, & Farrell, 1990), stability and dissolution properties of solid glucagons/g-cyclodextrin powder (Matilainen et al., 2009), hydration effects on gluten dynamics (Calucci, Forte, Galleschi, Geppi, & Ghiringhelli, 2003), and structure and molecular mobility of soy glycinin in the solid-state (Kealley et al., 2008). Based on these and similar studies, it would be possible to probe the change in structure and dynamic features of protein molecules in MPC powder samples stored at various water activities for different times from resolved signals representing different functional groups. This information may then be related to the decline in solubility of MPC powder during storage. Two excitation methods are commonly used in solid-state 13C NMR for this purpose (i) cross polarization (CP) including variable contact time experiments and (ii) direct polarization (DP) or single pulse excitation. Both techniques are usually combined with magic angle spinning (MAS) in order to average out chemical shift anisotropy (CSA). In order to prevent rapid loss of magnetization through dipolar relaxation, high power decoupling is usually used
E. Haque et al. / Food Hydrocolloids 44 (2015) 66e70
in combination with MAS. In the case of CP, magnetization is transferred from protons to carbons at a rate that depends on local molecular mobility and whether there is a proton covalently attached to a carbon site. Loss of magnetization after CP is characterized by T1(r) type relaxation on the 100 mse10 ms timescale. For carbon sites that are rigid enough to be cross polarized, the rate at which magnetization is transferred and subsequent decay (due to relaxation) depend on the local molecular motion with more rigid molecular segments having faster cross polarization. By varying the cross polarization contact time and monitoring how it affects the signal intensities, it is therefore possible to obtain information about the relative rigidity of molecular segments. In direct polarization, all carbon sites are directly magnetized. However, relatively rigid sites usually have very long subsequent T1 relaxation times. This means that to avoid saturation, very long delays between successive excitation pulses need to be employed to observe these signals. However, for a modest recycle time (e.g. 3 s as used here), then signals from rigid domains are effectively suppressed leaving only signals from relatively mobile segments that are able to be observed. The main objective of this study is to get a better understanding of the underlying molecular mechanisms associated with the loss of solubility of MPC upon prolonged storage using NMR techniques. 2. Materials and methods Milk protein concentrate powder was supplied by Murray Goulburn Co-operative Co. Ltd. (Brunswick, Victoria, Australia). Analysis of the powder was carried out by the manufacturer using the method described in Australian standard 2300 (standards Australia 1995). This showed the composition on a dry weight basis to be proteins (82.4% w/w), lactose (4% w/w), fat (1.6% w/w), ash (7.3% w/w) and moisture content (mc) 5.5% w/w (measured in our laboratory). In order to determine the effect of storage on the change in molecular structure and dynamics of MPC powder, two samples were used for both cross polarization magic angle spinning (CPMAS) with variable contact times and direct polarization experiments. These were MPC (mc ~5.5%) stored below e 4 C prior to analysis (shown to have solubility properties similar to fresh MPC and therefore used as a ‘non-aged’ control) and MPC powder stored at water activity (aw) 0.85 (mc ~16.5%) in vacuum-sealed desiccators containing saturated potassium chloride solution for 14 weeks at room temperature (25 C) (aged MPC). To examine the effect of the plasticization by water on molecular mobility/rigidity of protein molecules/molecular segments, MPC powder was stored in desiccators containing saturated salt solutions for up to 21 days to equilibrate the samples at aw 0.23, 0.43 and 0.85. The salt solutions used for this purpose were potassium acetate, potassium carbonate and potassium chloride respectively. 2.1. Water activity, moisture content and solubility test The water activities of the samples were tested before NMR analysis using an AquaLab 3TE water activity meter (Decagon Devices. Inc., Pullman, WA). The sample cup was half filled with sample and inserted in the measuring chamber of the meter. The sample was allowed to equilibrate with the head space of the sealed chamber containing a mirror and means of detecting condensation on the mirror. Water activity was measured at the point of initial condensation on the mirror at 25 C. Measurements were carried out in triplicate. Moisture content (dry basis) of the samples was determined by the AOAC Official Method 927.05 (AOAC International, 2000). The solubility of MPC was determined by a modification of the method described by Anema et al. (2006). Duplicate 5% MPC solutions were prepared from stored samples using MilliQ water. The solutions
67
were continuously stirred using a mechanical stirrer (rotor speed 400 rpm) at room temperature for 30 min. Sample solutions (40 mL) were transferred into 50 mL centrifugation tubes and were centrifuged at 1000 g for 10 min at 20 C. The supernatant was then filtered under vacuum (GF/A microfiber filter paper, 1.6 mm pore size, Whatman) and duplicate of 5 g aliquots of the filtrate weighed into pre-weighed aluminum dishes containing ~20 g acid washed sand and dried at 105 C for 24 h. The sample dishes were cooled to room temperature in a desiccator containing dry silica gel prior to weighing. The solubility of the MPC sample was calculated using the following equation:
% Solubility ¼
Solids in the supernatant 100 Solids in the solution
2.1.1. Statistical analysis Statistical significance of the change in solubility between different sample groups (aged and non-aged and equilibrated to different aw) was determined by calculating one-way ANOVA (analysis of variance) using the software Minitab 15, Minitab Inc., USA. 2.2.
13
C NMR experiments
The NMR measurements were performed using a Bruker MSL300 spectrometer operating at a frequency of 300.13 MHz for 1H and 75.482 MHz for 13C. The spectrometer was equipped with a 4mm double air bearing, magic angle spinning probe for MAS experiments. The 90 pulse time used in the high power protondecoupled single pulse 13C spectra was 5.7 ms. The spectrum width was 50 kHz, and 5000 data points were acquired. All samples were equilibrated at room temperature (~22 C). For variable contact time experiments, contact times were varied from 20 to 16,000 micro-seconds. In the case of DP (single pulse with high power decoupling) experiments, data acquisition parameters were number of scan 2000, acquisition time 35 ms and recycle delay 3 s. The sequence used for decoupling was Spinal 32. 3. Results and discussion 3.1. Change in solubility A steady decline in MPC solubility is observed (Table 1) upon storage of sample with increasing aw condition at ambient (25 C). After only 3 weeks of storage solubility decreased from 70% (nonaged) to around 29% for sample stored at aw 0.85 (Table 1). After 11 weeks of storage at high aw (0.85), solubility drop to only 7.5%. This result indicates that MPC solubility is greatly affected by storage condition such as water activity as well as storage period. 3.2. Storage effect on molecular structure and dynamics Fig. 1 shows typical cross polarization spectra for non-aged and aged (mc 16.6% w/w) MPC samples. Major resolved bands of the Table 1 Moisture content and solubility of MPC at different aw and storage period. MPC sample
Moisture content (w/w db)
Non-aged aw 0.23e3 wk aw 0.43e3 wk aw 0.85e3 wk aw 0.85e11 wk
5.5 5.4 8.2 16.4 16.6
a b
Non-significant p > 0.05. Significant p < 0.05.
± ± ± ± ±
0.1 0.3 0.1 0.6 0.2
Solubility (%) 70 65 48 29.6 7.5
± ± ± ± ±
1.3a 0.8a 2.2b 0.1b 2.6b
68
E. Haque et al. / Food Hydrocolloids 44 (2015) 66e70
spectra are for backbone carbonyl (~172 ppm), backbone a carbon (~54 ppm) and bands around 15e40 ppm which can be assigned to side chain methyl and methylene carbons (Kakalis et al., 1990). No significant difference in band position/intensity was observed between aged and non-aged samples which indicate that any structural change during ageing under high RH conditions did not have a marked effect on either chemical shift or relative intensity parameters. However, after storage, resonances were slightly more resolved particularly in the 20e60 ppm region. This suggests that storage results in a slightly better-defined set of local conformations. Variable contact time experiments were performed on these samples to investigate any change in relative rigidity/mobility of molecular segments/domains due to ageing. Fig. 2 compares intensity as a function of contact time for the major backbone peaks (172 and 53 ppm) of both aged and non-aged samples. For resonances due to chemically similar sites (such as backbone carbonyl or a-CH) experiencing similar local environments, a smooth increase in intensity due to magnetization build-up followed by a smooth decay due to relaxation was observed (Fig. 2) for both aged and non-aged samples. However a slight faster magnetization buildup was observed for MPC non-aged sample than the highmoisture-containing aged sample (Fig. 2) for the resolved bands at 172 and 54 ppm. Other bands at 20e40 ppm region also show a similar trend (data not shown). This may indicates that the rigidity of relatively immobile (i.e. observable in a CP spectrum) protein molecular segments (backbone as well as side chain) is higher for non-aged samples than the samples stored at aw 0.85 for a long period. This lower rigidity of molecular domains of aged samples could be due to plasticization by water or the effect of ageing or a combination of both. This will be further discussed later. Direct polarization (single pulse excitation) spectra (Fig. 3) of the same samples show that intensities of signals across the spectrum (backbone as well as side chain carbons) for non-aged MPC were similar to those of samples stored at aw 0.85 for 14 weeks. This period is sufficient for substantial loss of solubility (70% and 7.5% for non-aged and aged at aw 0.85 for 11 weeks respectively) of MPC. This result indicates that mobility (at 10e100 ps time-scale) of relatively mobile (i.e. observable in a DP spectrum) molecular segments did not change after storage of MPC powder at high humidity for a long time. No direct correlation between NMR parameters and MPC solubility is found and it could be due to the fact that NMR responds to the bulk proteins whereas loss of solubility is related to changes in limited amount of the protein e.g. surfaces of powder particles as revealed from a recent study (Mimouni, Deeth, Whittaker, Gidley, & Bhandari, 2010).
Fig. 1. CPMAS spectra of MPC (unequlibrated and stored at aw 0.85 for 14 weeks at 25 C temperature) for a 500 ms contact time. * ¼ spinning side band (peak assignments according to Kakalis et al. 1990).
Fig. 2. Relative intensities for (a) backbone carbonyl (172 ppm) and (b) a-CH (53 ppm) band resonance at variable contact time for non-aged (kept at-4 C) and aged samples stored at aw 0.85 for 14 weeks at 25 C.
3.3. Effect of plasticization Fig. 4 shows the effect of plasticization on the contact time vs. intensity plot for major resolved bands at 172 and 54 ppm for samples aged at different water activities. In both cases a smooth magnetization buildup followed by smooth decay was observed irrespective to the moisture content of the samples. A similar trend was observed for other resolved bands at 20e40 ppm (data not shown). This pattern of magnetization buildup and subsequent decay (contact time vs. intensity) remained very similar for samples containing moisture from 5.5 to around 17% (w/w). No difference in magnetization buildup and decay pattern was observed for any of the major resolved bands. This feature indicates that with increasing moisture content, protein molecules/molecular domains do not undergo structural change or the rigidity of the molecular segments do not change, at least at this (100 mse1 ms) timescale. For all moisture-containing samples including aged and non-aged samples (Fig. 2), carbonyl resonances experience the slowest buildup of magnetization, due to the absence of covalently attached H atoms. Direct polarization spectra also show no significant difference in band position and relative intensity between samples containing
E. Haque et al. / Food Hydrocolloids 44 (2015) 66e70
69
molecules due to plasticization during ageing which could facilitate proteineprotein interaction leading to the formation of a monolayer of fused casein micelles on the surface of the powder particles as suggested from electron microscopy study (Mimouni et al., 2010). An increase in molecular mobility in the bulk of the protein is therefore proposed to allow the formation of a surface ‘skin’ that slows down subsequent dissociation of micelles from powder particles, thereby resulting in low solubility. 4. Conclusion
Fig. 3. DP spectra of non-aged (kept at-4 C) and aged (kept at aw 0.85 for 14 weeks at 25 C temperature) MPC samples. * indicate spinning side band.
different moisture levels (from 5.5 to 17% w/w; data not shown) indicating molecular domains experiencing similar environments may not undergo change via interaction with water molecules. Previous studies on wheat gluten protein by CP experiments suggest hydration-induced mobility increase of molecular segments (aliphatic side chains) whereas an appreciable fraction of rigid gluten was still present after hydration (Calucci et al., 2003). An analogous result was obtained by Alberti et al. on hydration behaviour of high-molecular-weight wheat glutenin proteins (Alberti, Gilert, Tatham, Shewry, & Gil, 2002). Other recent studies on soy glycinin by cross polarization with the variable contact time technique show that at lower moisture contents (up to 17% w/w) a smooth buildup of magnetization and subsequent decay did not occur whereas for higher mc (~20e30% w/w) samples this conventional behaviour was observed (Kealley et al., 2008). This was explained as evidence of the presence of local domains of mobility with characteristically different magnetization buildup and decay rates (Kealley et al., 2008) for lower moisture containing samples. Our previous studies on watereprotein interaction (Haque et al., 2010) using T2 relaxometry showed that as water activity (i.e. moisture content) increases, water populations close to the protein surface also increase, i.e. watereprotein interactions increased which may favour protein denaturation. Also our studies on enthalpy relaxation or b-relaxation (which is usually associated with vibration and/or reorientation of side groups of polymer chains (Liu, Bhandari, & Zhou, 2006)) of MPC upon storage at increasing RH conditions showed increased enthalpy relaxation with increasing moisture content and storage period (Haque et al., 2012). The similar time-scale of enthalpy relaxation and solubility loss of MPC suggests that enthalpy relaxation may facilitate proteineprotein interaction. It was expected that increased watereprotein interaction would affect the relative mobility of molecular domains which is not obvious from the results of the current study. However variable contact time experiment data for non-aged MPC and MPC aged at high relative humidity (RH) (mc 16.5%) for a long period (14 weeks) showed a faster magnetization buildup and subsequent decay for the non-aged sample compared to that of the high-moisture-containing long-aged sample. This indicates higher rigidity of molecular domains of non-aged MPC compared to that of the long aged (at 25 C) MPC. It could be suggested from this observation that long-term storage at high RH may reduce rigidity of molecular domains due to interaction with water rather than short-term storage at high RH. This is consistent with increased molecular mobility of backbone and side chains of protein
It was found that a slight shorter time was required for non-aged MPC samples to magnetization buildup and subsequent decay compared to that of long-aged (at high aw) samples. This may indicate that the rigidity of molecular segments of protein in nonaged MPC was higher than the samples stored at high aw for long periods. This observation suggests that long-term storage at high RH may reduce rigidity of molecular domains due to interaction with water rather than short-term storage at high RH. Also the effect of plasticization (by water) studies suggest that with
Fig. 4. Relative intensities for (a) backbone carbonyl (172 ppm) and (b) a-CH (53 ppm) band resonance at variable contact time for samples stored at aw 0.23, 0.45 and 0.85 (moisture content 5.5, 8.2, 16.5 % w/w) for 21days at 25 C.
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E. Haque et al. / Food Hydrocolloids 44 (2015) 66e70
increasing moisture content protein molecules/molecular segments do not undergo any structural change or change in rigidity upon short-term storage. This study gives insight into the change in molecular structure and dynamics associated with the gradual loss of solubility of MPC during prolonged shelf life. Acknowledgement This research was supported by Dairy Innovation Australia Limited and the Australian Research Council (ARC) through ARC Linkage Grant No LP0669191 and ARC Linkage Infrastructure Grants No LE0775684 and LE0668517. References Alberti, E., Gilert, S. M., Tatham, A. S., Shewry, P. R., & Gil, A. M. (2002). Study of high molecular weight wheat glutenin subunit 1Dx5 by C-13 and H-1 solid-state NMR spectroscopy. I. Role of covalent crosslinking. Biopolymers, 67(6), 487e498. Anema, S. G., Pinder, D. N., Hunter, R. J., & Hemar, Y. (2006). Effects of storage temperature on the solubility of milk protein concentrate (MPC85). Food Hydrocolloids, 20(2e3), 386e393. Baldus, M. (2007). ICMRBS founder's medal 2006: biological solid-state NMR, methods and applications. Journal of Biomolecular NMR, 39(1), 73e86. Belloque, J., & Ramos, M. (1999). Application of NMR spectroscopy to milk and dairy products. Trends in Food Science & Technology, 10(10), 313e320. Calucci, L., Forte, C., Galleschi, L., Geppi, M., & Ghiringhelli, S. (2003). C-13 and H-1 solid state NMR investigation of hydration effects on gluten dynamics. International Journal of Biological Macromolecules, 32(3e5), 179e189.
Haque, E., Bhandari, B. R., Gidley, M. J., Deeth, H. C., Moller, S. M., & Whittaker, A. K. (2010). Protein conformational modifications and kinetics of water-protein interactions in milk protein concentrate powder upon aging: effect on solubility. Journal of Agricultural and Food Chemistry, 58(13), 7748e7755. Haque, E., Whittaker, A. K., Gidley, M. J., Deeth, H. C., Fibrianto, K., & Bhandari, B. R. (2012). Kinetics of enthalpy relaxation of milk protein concentrate powder upon ageing and its effect on solubility. Food Chemistry, 134(3), 1368e1373. http://dx.doi.org/10.1016/j.foodchem.2012.03.034. Havea, P. (2006). Protein interactions in milk protein concentrate powders. International Dairy Journal, 16(5), 415e422. Kakalis, L. T., Kumosinski, T. F., & Farrell, H. M. (1990). A multinuclear, highresolution NMR-study of bovine casein micelles and submicelles. Biophysical Chemistry, 38(1e2), 87e98. Kakalis, L. T., Kumosinski, T. F., & Farrell, H. M. (1994). The potential of solid-state nuclear-magnetic-resonance in dairy research e An application to cheese. Journal of Dairy Science, 77(3), 667e671. Kealley, C. S., Rout, M. k, Dezfouli, M. R., Strounina, E., Whittaker, A. K., Appelqvist, I. A. M., et al. (2008). Structure and molecular mobility of soy glycinin in the solid state. Biomacromolecules, 9(10), 2937e2946. Liu, Y. T., Bhandari, B., & Zhou, W. B. (2006). Glass transition and enthalpy relaxation of amorphous food saccharides: a review. Journal of Agricultural and Food Chemistry, 54(16), 5701e5717. Matilainen, L., Maunu, S. L., Pajander, J., Auriola, S., Jaaskelainen, I., Larsen, K. L., et al. (2009). The stability and dissolution properties of solid glucagon/gammacyclodextrin powder. European Journal of Pharmaceutical Sciences, 36(4e5), 412e420. Mimouni, A., Deeth, H. C., Whittaker, A. K., Gidley, M. J., & Bhandari, B. R. (2009). Rehydration process of milk protein concentrate powder monitored by static light scattering. Food Hydrocolloids, 23(7), 1958e1965. Mimouni, A., Deeth, H. C., Whittaker, A. K., Gidley, M. J., & Bhandari, B. R. (2010). Investigation of the microstructure of milk protein concentrate powders during rehydration: alterations during storage. Journal of Dairy Science, 93(2), 463e472.
Contents lists available at ScienceDirect
Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Change in molecular structure and dynamics of protein in milk protein concentrate powder upon ageing by solid-state carbon NMR Enamul Haque a, *, Bhesh R. Bhandari b, Michael J. Gidley c, Hilton C. Deeth b, Andrew K. Whittaker d a
School of Applied Sciences, RMIT University, VIC 3000, Australia School of Agriculture and Food Sciences, University of Queensland, QLD 4072 Australia Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, University of Queensland, QLD 4072, Australia d Centre for Advanced Imaging and Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland, QLD 4072, Australia b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 May 2014 Accepted 9 September 2014 Available online 18 September 2014
Instability of proteins in dry form causes solubility loss of milk protein concentrate (MPC) powder upon ageing. High resolution solid state NMR techniques were used to investigate the changes in molecular structure and dynamics of proteins in MPC with varying moisture content (5.5e16.5% w/w) and storage period. The results indicate a slight higher rigidity of molecular domains of protein molecules of nonaged MPC compared to that of the long aged (at 25 C) MPC. It could be suggested from this observation that long-term storage at high relative humidity (RH) may reduce rigidity of the molecular domains due to interaction with water rather than short-term storage at high RH. This may indicate increased molecular mobility of backbone and side chains of protein molecules due to plasticization during ageing which could facilitate proteineprotein interaction and protein denaturation. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Solid-state carbon NMR Milk protein concentrate Water activity Molecular mobility Solubility
1. Introduction Milk protein concentrate (MPC) is a dairy-derived powder with high protein content, up to 85%. MPC powders are used as a food ingredient in a variety of applications for their high nutritional value and favourable functional properties. Generally, MPC powders exhibit a gradual loss of solubility upon storage (Anema, Pinder, Hunter, & Hemar, 2006; Haque et al., 2010; Havea, 2006; Mimouni, Deeth, Whittaker, Gidley, & Bhandari, 2009). Changes in the structure and dynamics of protein molecules/segments in MPC powder could occur upon storage and this could have some link with solubility loss of MPC upon ageing. Solid-state carbon NMR techniques can be used to study these changes in the structure and dynamic features of protein molecules in MPC powder after storage at different conditions. This will provide an insight into the underlying molecular mechanism of protein aggregation that is most likely responsible for the loss of solubility on storage of MPC. Solid-state NMR has widespread applications in various fields including dairy and other food products (Baldus, 2007; Belloque &
* Corresponding author. E-mail address: [email protected] (E. Haque). http://dx.doi.org/10.1016/j.foodhyd.2014.09.022 0268-005X/© 2014 Elsevier Ltd. All rights reserved.
Ramos, 1999; Kakalis, Kumosinski, & Farrell, 1994). High resolution solid-state carbon NMR is a useful tool that can be used to monitor the mobility (or rigidity) of molecular segments in the backbone as well as side chains of protein molecules. This technique has been used to probe the calcium binding sites of casein and to ascertain the dynamic state of amino acid residues within casein micelles (Kakalis, Kumosinski, & Farrell, 1990), stability and dissolution properties of solid glucagons/g-cyclodextrin powder (Matilainen et al., 2009), hydration effects on gluten dynamics (Calucci, Forte, Galleschi, Geppi, & Ghiringhelli, 2003), and structure and molecular mobility of soy glycinin in the solid-state (Kealley et al., 2008). Based on these and similar studies, it would be possible to probe the change in structure and dynamic features of protein molecules in MPC powder samples stored at various water activities for different times from resolved signals representing different functional groups. This information may then be related to the decline in solubility of MPC powder during storage. Two excitation methods are commonly used in solid-state 13C NMR for this purpose (i) cross polarization (CP) including variable contact time experiments and (ii) direct polarization (DP) or single pulse excitation. Both techniques are usually combined with magic angle spinning (MAS) in order to average out chemical shift anisotropy (CSA). In order to prevent rapid loss of magnetization through dipolar relaxation, high power decoupling is usually used
E. Haque et al. / Food Hydrocolloids 44 (2015) 66e70
in combination with MAS. In the case of CP, magnetization is transferred from protons to carbons at a rate that depends on local molecular mobility and whether there is a proton covalently attached to a carbon site. Loss of magnetization after CP is characterized by T1(r) type relaxation on the 100 mse10 ms timescale. For carbon sites that are rigid enough to be cross polarized, the rate at which magnetization is transferred and subsequent decay (due to relaxation) depend on the local molecular motion with more rigid molecular segments having faster cross polarization. By varying the cross polarization contact time and monitoring how it affects the signal intensities, it is therefore possible to obtain information about the relative rigidity of molecular segments. In direct polarization, all carbon sites are directly magnetized. However, relatively rigid sites usually have very long subsequent T1 relaxation times. This means that to avoid saturation, very long delays between successive excitation pulses need to be employed to observe these signals. However, for a modest recycle time (e.g. 3 s as used here), then signals from rigid domains are effectively suppressed leaving only signals from relatively mobile segments that are able to be observed. The main objective of this study is to get a better understanding of the underlying molecular mechanisms associated with the loss of solubility of MPC upon prolonged storage using NMR techniques. 2. Materials and methods Milk protein concentrate powder was supplied by Murray Goulburn Co-operative Co. Ltd. (Brunswick, Victoria, Australia). Analysis of the powder was carried out by the manufacturer using the method described in Australian standard 2300 (standards Australia 1995). This showed the composition on a dry weight basis to be proteins (82.4% w/w), lactose (4% w/w), fat (1.6% w/w), ash (7.3% w/w) and moisture content (mc) 5.5% w/w (measured in our laboratory). In order to determine the effect of storage on the change in molecular structure and dynamics of MPC powder, two samples were used for both cross polarization magic angle spinning (CPMAS) with variable contact times and direct polarization experiments. These were MPC (mc ~5.5%) stored below e 4 C prior to analysis (shown to have solubility properties similar to fresh MPC and therefore used as a ‘non-aged’ control) and MPC powder stored at water activity (aw) 0.85 (mc ~16.5%) in vacuum-sealed desiccators containing saturated potassium chloride solution for 14 weeks at room temperature (25 C) (aged MPC). To examine the effect of the plasticization by water on molecular mobility/rigidity of protein molecules/molecular segments, MPC powder was stored in desiccators containing saturated salt solutions for up to 21 days to equilibrate the samples at aw 0.23, 0.43 and 0.85. The salt solutions used for this purpose were potassium acetate, potassium carbonate and potassium chloride respectively. 2.1. Water activity, moisture content and solubility test The water activities of the samples were tested before NMR analysis using an AquaLab 3TE water activity meter (Decagon Devices. Inc., Pullman, WA). The sample cup was half filled with sample and inserted in the measuring chamber of the meter. The sample was allowed to equilibrate with the head space of the sealed chamber containing a mirror and means of detecting condensation on the mirror. Water activity was measured at the point of initial condensation on the mirror at 25 C. Measurements were carried out in triplicate. Moisture content (dry basis) of the samples was determined by the AOAC Official Method 927.05 (AOAC International, 2000). The solubility of MPC was determined by a modification of the method described by Anema et al. (2006). Duplicate 5% MPC solutions were prepared from stored samples using MilliQ water. The solutions
67
were continuously stirred using a mechanical stirrer (rotor speed 400 rpm) at room temperature for 30 min. Sample solutions (40 mL) were transferred into 50 mL centrifugation tubes and were centrifuged at 1000 g for 10 min at 20 C. The supernatant was then filtered under vacuum (GF/A microfiber filter paper, 1.6 mm pore size, Whatman) and duplicate of 5 g aliquots of the filtrate weighed into pre-weighed aluminum dishes containing ~20 g acid washed sand and dried at 105 C for 24 h. The sample dishes were cooled to room temperature in a desiccator containing dry silica gel prior to weighing. The solubility of the MPC sample was calculated using the following equation:
% Solubility ¼
Solids in the supernatant 100 Solids in the solution
2.1.1. Statistical analysis Statistical significance of the change in solubility between different sample groups (aged and non-aged and equilibrated to different aw) was determined by calculating one-way ANOVA (analysis of variance) using the software Minitab 15, Minitab Inc., USA. 2.2.
13
C NMR experiments
The NMR measurements were performed using a Bruker MSL300 spectrometer operating at a frequency of 300.13 MHz for 1H and 75.482 MHz for 13C. The spectrometer was equipped with a 4mm double air bearing, magic angle spinning probe for MAS experiments. The 90 pulse time used in the high power protondecoupled single pulse 13C spectra was 5.7 ms. The spectrum width was 50 kHz, and 5000 data points were acquired. All samples were equilibrated at room temperature (~22 C). For variable contact time experiments, contact times were varied from 20 to 16,000 micro-seconds. In the case of DP (single pulse with high power decoupling) experiments, data acquisition parameters were number of scan 2000, acquisition time 35 ms and recycle delay 3 s. The sequence used for decoupling was Spinal 32. 3. Results and discussion 3.1. Change in solubility A steady decline in MPC solubility is observed (Table 1) upon storage of sample with increasing aw condition at ambient (25 C). After only 3 weeks of storage solubility decreased from 70% (nonaged) to around 29% for sample stored at aw 0.85 (Table 1). After 11 weeks of storage at high aw (0.85), solubility drop to only 7.5%. This result indicates that MPC solubility is greatly affected by storage condition such as water activity as well as storage period. 3.2. Storage effect on molecular structure and dynamics Fig. 1 shows typical cross polarization spectra for non-aged and aged (mc 16.6% w/w) MPC samples. Major resolved bands of the Table 1 Moisture content and solubility of MPC at different aw and storage period. MPC sample
Moisture content (w/w db)
Non-aged aw 0.23e3 wk aw 0.43e3 wk aw 0.85e3 wk aw 0.85e11 wk
5.5 5.4 8.2 16.4 16.6
a b
Non-significant p > 0.05. Significant p < 0.05.
± ± ± ± ±
0.1 0.3 0.1 0.6 0.2
Solubility (%) 70 65 48 29.6 7.5
± ± ± ± ±
1.3a 0.8a 2.2b 0.1b 2.6b
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spectra are for backbone carbonyl (~172 ppm), backbone a carbon (~54 ppm) and bands around 15e40 ppm which can be assigned to side chain methyl and methylene carbons (Kakalis et al., 1990). No significant difference in band position/intensity was observed between aged and non-aged samples which indicate that any structural change during ageing under high RH conditions did not have a marked effect on either chemical shift or relative intensity parameters. However, after storage, resonances were slightly more resolved particularly in the 20e60 ppm region. This suggests that storage results in a slightly better-defined set of local conformations. Variable contact time experiments were performed on these samples to investigate any change in relative rigidity/mobility of molecular segments/domains due to ageing. Fig. 2 compares intensity as a function of contact time for the major backbone peaks (172 and 53 ppm) of both aged and non-aged samples. For resonances due to chemically similar sites (such as backbone carbonyl or a-CH) experiencing similar local environments, a smooth increase in intensity due to magnetization build-up followed by a smooth decay due to relaxation was observed (Fig. 2) for both aged and non-aged samples. However a slight faster magnetization buildup was observed for MPC non-aged sample than the highmoisture-containing aged sample (Fig. 2) for the resolved bands at 172 and 54 ppm. Other bands at 20e40 ppm region also show a similar trend (data not shown). This may indicates that the rigidity of relatively immobile (i.e. observable in a CP spectrum) protein molecular segments (backbone as well as side chain) is higher for non-aged samples than the samples stored at aw 0.85 for a long period. This lower rigidity of molecular domains of aged samples could be due to plasticization by water or the effect of ageing or a combination of both. This will be further discussed later. Direct polarization (single pulse excitation) spectra (Fig. 3) of the same samples show that intensities of signals across the spectrum (backbone as well as side chain carbons) for non-aged MPC were similar to those of samples stored at aw 0.85 for 14 weeks. This period is sufficient for substantial loss of solubility (70% and 7.5% for non-aged and aged at aw 0.85 for 11 weeks respectively) of MPC. This result indicates that mobility (at 10e100 ps time-scale) of relatively mobile (i.e. observable in a DP spectrum) molecular segments did not change after storage of MPC powder at high humidity for a long time. No direct correlation between NMR parameters and MPC solubility is found and it could be due to the fact that NMR responds to the bulk proteins whereas loss of solubility is related to changes in limited amount of the protein e.g. surfaces of powder particles as revealed from a recent study (Mimouni, Deeth, Whittaker, Gidley, & Bhandari, 2010).
Fig. 1. CPMAS spectra of MPC (unequlibrated and stored at aw 0.85 for 14 weeks at 25 C temperature) for a 500 ms contact time. * ¼ spinning side band (peak assignments according to Kakalis et al. 1990).
Fig. 2. Relative intensities for (a) backbone carbonyl (172 ppm) and (b) a-CH (53 ppm) band resonance at variable contact time for non-aged (kept at-4 C) and aged samples stored at aw 0.85 for 14 weeks at 25 C.
3.3. Effect of plasticization Fig. 4 shows the effect of plasticization on the contact time vs. intensity plot for major resolved bands at 172 and 54 ppm for samples aged at different water activities. In both cases a smooth magnetization buildup followed by smooth decay was observed irrespective to the moisture content of the samples. A similar trend was observed for other resolved bands at 20e40 ppm (data not shown). This pattern of magnetization buildup and subsequent decay (contact time vs. intensity) remained very similar for samples containing moisture from 5.5 to around 17% (w/w). No difference in magnetization buildup and decay pattern was observed for any of the major resolved bands. This feature indicates that with increasing moisture content, protein molecules/molecular domains do not undergo structural change or the rigidity of the molecular segments do not change, at least at this (100 mse1 ms) timescale. For all moisture-containing samples including aged and non-aged samples (Fig. 2), carbonyl resonances experience the slowest buildup of magnetization, due to the absence of covalently attached H atoms. Direct polarization spectra also show no significant difference in band position and relative intensity between samples containing
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molecules due to plasticization during ageing which could facilitate proteineprotein interaction leading to the formation of a monolayer of fused casein micelles on the surface of the powder particles as suggested from electron microscopy study (Mimouni et al., 2010). An increase in molecular mobility in the bulk of the protein is therefore proposed to allow the formation of a surface ‘skin’ that slows down subsequent dissociation of micelles from powder particles, thereby resulting in low solubility. 4. Conclusion
Fig. 3. DP spectra of non-aged (kept at-4 C) and aged (kept at aw 0.85 for 14 weeks at 25 C temperature) MPC samples. * indicate spinning side band.
different moisture levels (from 5.5 to 17% w/w; data not shown) indicating molecular domains experiencing similar environments may not undergo change via interaction with water molecules. Previous studies on wheat gluten protein by CP experiments suggest hydration-induced mobility increase of molecular segments (aliphatic side chains) whereas an appreciable fraction of rigid gluten was still present after hydration (Calucci et al., 2003). An analogous result was obtained by Alberti et al. on hydration behaviour of high-molecular-weight wheat glutenin proteins (Alberti, Gilert, Tatham, Shewry, & Gil, 2002). Other recent studies on soy glycinin by cross polarization with the variable contact time technique show that at lower moisture contents (up to 17% w/w) a smooth buildup of magnetization and subsequent decay did not occur whereas for higher mc (~20e30% w/w) samples this conventional behaviour was observed (Kealley et al., 2008). This was explained as evidence of the presence of local domains of mobility with characteristically different magnetization buildup and decay rates (Kealley et al., 2008) for lower moisture containing samples. Our previous studies on watereprotein interaction (Haque et al., 2010) using T2 relaxometry showed that as water activity (i.e. moisture content) increases, water populations close to the protein surface also increase, i.e. watereprotein interactions increased which may favour protein denaturation. Also our studies on enthalpy relaxation or b-relaxation (which is usually associated with vibration and/or reorientation of side groups of polymer chains (Liu, Bhandari, & Zhou, 2006)) of MPC upon storage at increasing RH conditions showed increased enthalpy relaxation with increasing moisture content and storage period (Haque et al., 2012). The similar time-scale of enthalpy relaxation and solubility loss of MPC suggests that enthalpy relaxation may facilitate proteineprotein interaction. It was expected that increased watereprotein interaction would affect the relative mobility of molecular domains which is not obvious from the results of the current study. However variable contact time experiment data for non-aged MPC and MPC aged at high relative humidity (RH) (mc 16.5%) for a long period (14 weeks) showed a faster magnetization buildup and subsequent decay for the non-aged sample compared to that of the high-moisture-containing long-aged sample. This indicates higher rigidity of molecular domains of non-aged MPC compared to that of the long aged (at 25 C) MPC. It could be suggested from this observation that long-term storage at high RH may reduce rigidity of molecular domains due to interaction with water rather than short-term storage at high RH. This is consistent with increased molecular mobility of backbone and side chains of protein
It was found that a slight shorter time was required for non-aged MPC samples to magnetization buildup and subsequent decay compared to that of long-aged (at high aw) samples. This may indicate that the rigidity of molecular segments of protein in nonaged MPC was higher than the samples stored at high aw for long periods. This observation suggests that long-term storage at high RH may reduce rigidity of molecular domains due to interaction with water rather than short-term storage at high RH. Also the effect of plasticization (by water) studies suggest that with
Fig. 4. Relative intensities for (a) backbone carbonyl (172 ppm) and (b) a-CH (53 ppm) band resonance at variable contact time for samples stored at aw 0.23, 0.45 and 0.85 (moisture content 5.5, 8.2, 16.5 % w/w) for 21days at 25 C.
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increasing moisture content protein molecules/molecular segments do not undergo any structural change or change in rigidity upon short-term storage. This study gives insight into the change in molecular structure and dynamics associated with the gradual loss of solubility of MPC during prolonged shelf life. Acknowledgement This research was supported by Dairy Innovation Australia Limited and the Australian Research Council (ARC) through ARC Linkage Grant No LP0669191 and ARC Linkage Infrastructure Grants No LE0775684 and LE0668517. References Alberti, E., Gilert, S. M., Tatham, A. S., Shewry, P. R., & Gil, A. M. (2002). Study of high molecular weight wheat glutenin subunit 1Dx5 by C-13 and H-1 solid-state NMR spectroscopy. I. Role of covalent crosslinking. Biopolymers, 67(6), 487e498. Anema, S. G., Pinder, D. N., Hunter, R. J., & Hemar, Y. (2006). Effects of storage temperature on the solubility of milk protein concentrate (MPC85). Food Hydrocolloids, 20(2e3), 386e393. Baldus, M. (2007). ICMRBS founder's medal 2006: biological solid-state NMR, methods and applications. Journal of Biomolecular NMR, 39(1), 73e86. Belloque, J., & Ramos, M. (1999). Application of NMR spectroscopy to milk and dairy products. Trends in Food Science & Technology, 10(10), 313e320. Calucci, L., Forte, C., Galleschi, L., Geppi, M., & Ghiringhelli, S. (2003). C-13 and H-1 solid state NMR investigation of hydration effects on gluten dynamics. International Journal of Biological Macromolecules, 32(3e5), 179e189.
Haque, E., Bhandari, B. R., Gidley, M. J., Deeth, H. C., Moller, S. M., & Whittaker, A. K. (2010). Protein conformational modifications and kinetics of water-protein interactions in milk protein concentrate powder upon aging: effect on solubility. Journal of Agricultural and Food Chemistry, 58(13), 7748e7755. Haque, E., Whittaker, A. K., Gidley, M. J., Deeth, H. C., Fibrianto, K., & Bhandari, B. R. (2012). Kinetics of enthalpy relaxation of milk protein concentrate powder upon ageing and its effect on solubility. Food Chemistry, 134(3), 1368e1373. http://dx.doi.org/10.1016/j.foodchem.2012.03.034. Havea, P. (2006). Protein interactions in milk protein concentrate powders. International Dairy Journal, 16(5), 415e422. Kakalis, L. T., Kumosinski, T. F., & Farrell, H. M. (1990). A multinuclear, highresolution NMR-study of bovine casein micelles and submicelles. Biophysical Chemistry, 38(1e2), 87e98. Kakalis, L. T., Kumosinski, T. F., & Farrell, H. M. (1994). The potential of solid-state nuclear-magnetic-resonance in dairy research e An application to cheese. Journal of Dairy Science, 77(3), 667e671. Kealley, C. S., Rout, M. k, Dezfouli, M. R., Strounina, E., Whittaker, A. K., Appelqvist, I. A. M., et al. (2008). Structure and molecular mobility of soy glycinin in the solid state. Biomacromolecules, 9(10), 2937e2946. Liu, Y. T., Bhandari, B., & Zhou, W. B. (2006). Glass transition and enthalpy relaxation of amorphous food saccharides: a review. Journal of Agricultural and Food Chemistry, 54(16), 5701e5717. Matilainen, L., Maunu, S. L., Pajander, J., Auriola, S., Jaaskelainen, I., Larsen, K. L., et al. (2009). The stability and dissolution properties of solid glucagon/gammacyclodextrin powder. European Journal of Pharmaceutical Sciences, 36(4e5), 412e420. Mimouni, A., Deeth, H. C., Whittaker, A. K., Gidley, M. J., & Bhandari, B. R. (2009). Rehydration process of milk protein concentrate powder monitored by static light scattering. Food Hydrocolloids, 23(7), 1958e1965. Mimouni, A., Deeth, H. C., Whittaker, A. K., Gidley, M. J., & Bhandari, B. R. (2010). Investigation of the microstructure of milk protein concentrate powders during rehydration: alterations during storage. Journal of Dairy Science, 93(2), 463e472.