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Physicochemical properties of skim milk powder dispersions after acidification to pH 2.4–3.0 and heating
Food Hydrocolloids 100 (2020) 105435
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Physicochemical properties of skim milk powder dispersions after acidification to pH 2.4–3.0 and heating Inseob Choi, Qixin Zhong * Department of Food Science, The University of Tennessee, Knoxville, TN, 37996, United States
A R T I C L E I N F O
A B S T R A C T
Keywords: Skim milk powder dispersion Acidification Heating Casein micelle Colloidal calcium phosphate Hydrophobic interaction
Application of skim milk powder (SMP) in acidified beverages is limited due to precipitation and sedimentation of caseins. In this study, 5% w/v SMP dispersion was acidified to pH 2.4–3.0 using citric acid and heated at different conditions. Lowering pH resulted in dissolution of colloidal calcium phosphate from casein micelles, corresponding to lowered turbidity and reduced hydrodynamic diameter of dispersions. When heated for 10 min at different temperatures, 70 � C was the most effective to produce transparent dispersions; whereas a shorter heating time between 2 and 60 min was desired at 90 � C. The decreased turbidity after heating was attributed to weakened hydrophobic interaction between casein molecules based on fluorescence spectrometry and ion analysis. Scanning transmission electron microscopy showed the altered structure and decreased dimension of casein micelles after acidification and heating. The current study may expand the use of SMP for manufacturing new types of protein drinks.
1. Introduction Skim milk powder (SMP) contains high-quality proteins and a fat content of less than 1.2% wt (Devine, Bell, & Prince, 1996). Due to its long shelf-life and high protein content, SMP has been used in a wide range of applications including reconstituted and recombined dairy products (Anema & McKenna, 1996), confectionery (Liang & Hartel, €hko €nen, Tuorila, & Hyvo €nen, 1995), and diet beverages 2004), soups (Ka (Mesirow & Welsh, 2015). Casein micelles constitute 80% of milk pro teins and play an important role to determine physicochemical proper ties of SMP dispersions; the other 20% milk proteins are whey proteins. Despite nutritional quality of SMP, the precipitation of casein micelles limits the use of SMP in acidic beverages without hydrocolloid stabi lizers. Additionally, beverages with lowered turbidity are perceived by consumers as healthier, natural, and less contaminated products (Tse & Yim, 2002). Studies are therefore needed to dissociate casein micelles to reduce turbidity of and stabilize caseins in acidified beverages. The structure of casein micelles is enabled by a balance between attractive hydrophobic and repulsive electrostatic interactions, as well as colloidal calcium phosphate (CCP) bridging calcium sensitive ɑs1-and β-caseins that are highly phosphorylated (Liu & Guo, 2008). The CCP bridging strengthens the short-range hydrophobic interactions between hydrophobic regions of caseins and neutralizes negatively charged
phosphoserine groups (Liu et al., 2008). The knowledge of interactions in casein micelles can be used to guide studies to reduce milk turbidity. CCP is in equilibrium with free calcium and phosphate ions in the serum phase of milk (Walstra, 1999), and chelating agents with a stronger binding affinity to calcium than phosphorylated caseins result in the dissolving of CCP and therefore dissociation of casein micelles (Kaliap pan & Lucey, 2011; Lazzaro et al., 2017). The dissociation of casein micelles has also been reported after high-pressure treatments that disrupt CCP linkages with casein (Bravo, Felipe, Lopez-Fandino, & Molina, 2013). Acidification from pH 6.0 to pH 4.0 results in the increased concentrations of calcium and phosphate in milk serum, suggesting the dissolving of CCP in casein micelles (Le Gra€et & Gau cheron, 1999); acidification however weakens electrostatic repulsion to cause aggregation of caseins. Thermal treatment is used to ensure the safety and shelf-life of milk. When skim milk is heated, heterogeneous casein-whey protein com plexes can be formed with whey proteins depositing onto casein micelles (Dalgleish, van Mourik, & Corredig, 1997). Heating milk at 70–90 � C causes the denaturation and exposure of the buried reactive thiol groups of beta-lactoglobulin (β-Lg) (Le et al., 2007) to form disulfide bonds between β-Lg and κ-casein on casein micelle surface by thiol-disulfide interchange reactions (Jang & Swaisgood, 1990). This has been shown for the maintained particle size distribution of milk depleted for whey
* Corresponding author. E-mail address: [email protected] (Q. Zhong). https://doi.org/10.1016/j.foodhyd.2019.105435 Received 24 July 2019; Received in revised form 10 October 2019; Accepted 10 October 2019 Available online 14 October 2019 0268-005X/© 2019 Elsevier Ltd. All rights reserved.
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proteins after heating (Le, Saveyn, Hoa, & Van der Meeren, 2008). The extent of association between whey proteins and casein micelles is greater at a lower pH in the range from 7.3 to 5.8 (Smits & van Brou wershaven, 1980). Despite popularity in Europe and Asia, acidified milk products are susceptible to sedimentation due to casein aggregation around pH 3.8–5.0 (Amice-Quemeneur, Haluk, Hardy, & Kravtchenko, 1995). Regardless of differences in the composition of casein micelles and mineral balance in skim milk, gelation occurs when milk is subjected to acidification (e.g., ~pH 4.2) using glucono-δ-lactone (GDL) (Ould Eleya, Desobry Banon, & Hardy, 1995). Additionally, the traditional acidifi cation method using GDL has poor reproducibility (Amice-Quemeneur et al., 1995). Physicochemical properties of skim milk and casein mi celles below pH 3.8 have not been reported but are significant to many acidic beverages that can be potentially fortified with casein-based dairy proteins. The overall objective of this study was to reduce the turbidity of SMP dispersions at pH 2.4–3.0 and characterize physicochemical properties of SMP dispersions after acidification with citric acid and heating. Citric acid was used as it is commonly used in food beverage production and citrate is a well-known chelating agent. The fundamental information was studied to understand turbidity changes of SMP dispersions using dynamic light scattering (DLS), fluorescence spectrometry, inductively coupled argon plasma-optical emission spectrometry (ICP-OES), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), freethiol (SH) group concentrations, and scanning transmission electron microscopy (STEM).
used to measure the particle size distribution of dispersions (model Zetasizer nano-ZS, Malvern Instrument, Worcestershire, UK). The mean hydrodynamic diameter (Dh) was calculated using Zetasizer Software 7.11. Measurements were done with a backscatter angle of 173� and an equilibrium temperature of 25 � C. 2.4. Scanning transmission electron microscopy The morphology of particulates before and after acidification and after heating at 90 � C for 2 min was studied using STEM. The 5% w/v SMP dispersions were diluted five times with the corresponding serum obtained using the centrifugal ultrafiltration method detailed in the below ICP-OES section. The diluted 1% w/v SMP dispersion was placed on a piece of Parafilm. A freshly glow discharged 400 mesh copper grid with a thin carbon film was placed on the sample drop. After 1 min, excess sample was quickly removed from the grid and the grid was placed on a drop of water for 10 s. The water was removed using Kim wipes™ and the grid was placed on a drop of 1% uranyl acetate for 1 min. Then excess stain was quickly removed using Kimwipes™ and the sample was allowed to dry at RT. The stained samples were imaged using a Zeiss Auriga microscope (Carl Zeiss, Thornwood, NY) operating in the STEM mode at 30 kV. 2.5. Fluorescence spectrometry Steady state fluorescence spectra were measured using a LS 55 fluorescence spectrometer (PerkinElmer, Waltham, MA) to study effects of acidification and heating on the structure of proteins in SMP disper sions. To acquire intrinsic fluorescence spectra, the excitation wave length was 295 nm and the range of emission spectra was between 300 and 500 nm. Pyrene and 8-anilinonaphthalene-1-sulfonic acid (ANS) were used individually at 1.0 � 10 6 and 1.0 � 10 5 M, respectively, in 5% w/v SMP dispersions, as probes binding to hydrophobic sites of proteins, and the spectra were obtained at an excitation wavelength of 338 and 380 nm, respectively. The emission spectra were collected from 350 to 600 nm for SMP dispersions with pyrene and from 400 to 560 nm for those with ANS (Liu et al., 2008). Excitation and emission slit sizes were fixed at 5.0 nm for intrinsic fluorescence spectra and pyrene treatments and 2.5 nm for ANS treatments. The scan rate for all mea surements was set as 240 nm/min.
2. Materials and methods 2.1. Materials Carnation® SMP (Nestle� USA, Solon, OH) was used in the present study. The SMP was determined to contain 35.4% protein based on the Kjeldahl method (AOAC, 2005). The calcium content in SMP was determined to be 2.10% after dry ashing and ICP-OES detailed below. Citric acid was purchased from Fisher Scientific (Fair Lawn, NJ). Micellar casein (MC) was from American Casein Company (Burlington, NJ) and whey protein isolate (WPI) was from Hilmar Ingredients (Hil mar, CA). Glycine and Ellman’s reagent were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Tris, ethylenediaminetetraacetic acid, and urea were procured from Acros Organics (Pittsburgh, PA).
2.6. Inductively coupled plasma-optical emission spectrometry
2.2. Sample preparation
To investigate the effect of acidification and heating on the disso lution of CCP, acidified SMP dispersions were determined for serum calcium and phosphorous concentrations before and after heating at 90 � C for 2 min. The Amicon ultra-15 centrifugal filter unit having an ultracel-10 membrane with a molecular weight cut-off of 10,000 Da (Merck Millipore Ltd, Cork, Ireland) was used to obtain the serum phase of SMP dispersions. Centrifugation was performed at 4500 g for 3 h using Sorvall RC-5B plus centrifuge (Sorvall, Newtown, CT). Each serum was diluted 100 times with deionized water and analyzed using a Spectro Ciros ICP-OES instrument (Spectro Ciros, Mahwah, NJ). To calibrate the instrument before calcium and phosphorous analysis, ICP-OES standards were used (VHG Labs, Manchester, NH). The detection limit for calcium and phosphorous in the method was both 0.01 mg/L.
SMP was hydrated with deionized water at 5% w/v in 20 mL scin tillation vials. After vortexing, samples were incubated at room tem perature (RT, ~21 � C) for at least 8 h for complete hydration. Citric acid solution (2.0 M) was added drop-wise in the SMP dispersions until reaching pH 2.4, 2.7, and 3.0. The SMP dispersion at pH 6.8 without acidification was used as a control. To study interactions between casein micelles and whey proteins, dispersions with 1.73% w/v MC or 1.39% w/v MC and 0.39% w/v WPI, simulating the proximate mass ratio of casein and whey protein (4:1) in bovine milk (Zadow, 1994), were similarly prepared. Vials were incubated in a water bath pre-equilibrated to 60, 70, 80, and 90 � C for 2, 5, 10, 30, and 60 min. Thermal profiles of sample centers are shown in supporting Fig. S1 to guide future scale-up attempts. As the thermal come-up time varied, the treatments are still named at the set temperature for convenience in description.
2.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SMP dispersions without or with heating at 90 � C for 2 min were analyzed for both reducing and non-reducing SDS-PAGE. The sample buffer was prepared with β-mercaptoethanol and Laemmli sample buffer at a volume ratio of 5:95 for reducing SDS-PAGE, and β-mercaptoethanol was excluded for non-reducing SDS-PAGE. Each SMP dispersion was diluted 40 times with the sample buffer. Samples were heated in boiling water for 5 min before reducing SDS-PAGE. The running buffer for gel
2.3. Turbidity and particle size distribution Turbidity of SMP dispersions was measured at RT using a 2020 we/ wi turbidimeter (LaMotte, Chestertown, MD). Calibration was per formed using the AMCO primary turbidity standard (10 NTU). DLS was 2
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electrophoresis was prepared with 100 mL of 10� Tris/Glycine/SDS and 900 mL of deionized water. Ten microliters of a sample were loaded in each well of 4–20% Mini-PROTEAN TGX Gels (Bio-Rad Laboratories, Hercules, CA). The electrophoresis was run at 300 mA and 150 V for 2 h. After washing with deionized water three times for 5 min, the gels were stained using Bio-safe Coomassie G-250 Stain for 1 h and then rinsed with deionized water for 30 min. 2.8. Determination of free thiol (SH) group concentration Free thiol (SH) groups in SMP dispersions before and after acidifi cation and heating were determined using the method of Beveridge et al. (Beveridge, Toma, & Nakai, 1974) with slight modification. SMP dis persions prepared as above at pH 2.4, 2.7, or 3.0 were heated at 90 � C for 2 min. Two hundred microliters of a SMP dispersion were mixed in 1 mL of Tris-glycine buffer containing 0.086 M Tris, 0.09 M glycine, 4 mM ethylenediaminetetraacetic acid, and 8 M urea, and reacted with 8 μL of Ellman’s reagent containing 10 mM 5,50 -dithiobis (2-nitrobenzoic acid). After centrifugation at 10,000 g for 5 min (model 4540, Eppendorf, Hamburg, Germany), absorbance of supernatant was measured at 412 nm using a model Evolution 201 UV–Vis spectrophotometer (Thermo Scientific, Waltham, MA). The concentration of SH groups was determined using Beer’s law with the molar extinction coefficient of 13, 600 M 1cm 1. 2.9. Statistical analysis Three replicates of each sample on each test were prepared for sta tistical analysis. One-way analysis of variance (ANOVA) was performed using SAS enterprise guide 7.1 (SAS Institute Inc., Cary, NC). Mean and standard deviation (SD) were calculated and differences of calculated means were considered significant with P < 0.05 using Fisher’s least significance difference (LSD) test. 3. Results and discussion 3.1. Turbidity, hydrodynamic diameter, and particle size distribution of skim milk powder dispersions
Fig. 1. The appearance of 5% w/v SMP dispersions after acidification to pH 2.4–3.0 (a) and heating at 90 � C for 2 min (b), pH 2.4 and heating at 60–90 � C for 10 min (c), and pH 2.4 and heating at 90 � C for 2–30 min (d). The far-left sample is the unheated control at pH 6.8.
The appearance, turbidity and mean Dh, and particle size distribution of skim milk powder dispersions after various treatments are presented in Figs. 1–3, respectively. The results are presented according to the treatment variable.
study was to reduce SMP dispersion turbidity, the rest of heating treatments were studied at pH 2.4.
3.1.1. Effects of acidification Visual appearance of protein-containing beverages is an important quality parameter and is determined by the dimension, concentration, light refraction properties (refractive index), and stability of protein structures (Wagoner & Foegeding, 2017; Zhang & Reineccius, 2016). After acidification to a lower pH, SMP dispersions became visually more translucent (Fig. 1a). Correspondingly, the turbidity and Dh were smaller at a lower pH (Fig. 2a). All acidified dispersions showed a greater portion of particles smaller than the control, and the extent of casein micelle dissociation was greater at a lower pH (Fig. 3a). The less turbid appearance (Fig. 1a) but bigger Dh (Fig. 2a) of the pH 3.0 sample than the control at pH 6.8 suggest the aggregation of initially dissociated caseins. The results are in agreement with the observed casein micelle dissociation at pH 4.0 (Le Gra€ et et al., 1999). The re-aggregated casein particles may have a lower refractive index than casein micelles that are cross-linked by CCP, which causes less light scattering and a lower turbidity at pH 3.0 than at pH 6.8 (Griffin & Griffin, 1985). After heating at 90 � C for 2 min, similar to thermal treatment conditions (88–96 � C for 2 min) in the hot-fill process used to pasteurize high-acid beverages, all dispersions showed the decreases in turbidity and Dh (Fig. 2a vs. 2b) and the narrowed particle size distribution (Fig. 3b), and precipitation was observed for the pH 3.0 dispersion (Fig. 1b). Since the goal of the present
3.1.2. Effects of heating temperature When the pH 2.4 sample was heated for 10 min at 60–90 � C, turbidity of all samples decreased after heating (Fig. 2c vs. 2a), even in the sample heated at 60 � C which was more turbid than other samples (Figs. 1c and 2c). Heating dispersions at 70 � C resulted in the lowest turbidity and the smallest Dh (Fig. 2c). The particle size distributions were more mono dispersed after heating at 80 and 90 � C than at 60 and 70 � C (Fig. 3c), and there was no linear correlation between heating temperature and turbidity or Dh. The results in Fig. 2c suggests two different phenomena below and above 70 � C. The reported denaturation temperature of β-Lg is 75.9, 81.9, and 78.7 � C at pH 6.5, 3.5, and 2.5, respectively, while the respective denaturation temperature of whey protein mixtures at pH 6.5, 3.5, and 2.5 is 76.9, 88.0, and 80.6 � C (Bernal & Jelen, 1985). Above 70 � C, a positive relationship was reported between temperature and the amount of denatured whey proteins, leading to increases in turbidity and Dh of milk (Dannenberg & Kessler, 1988), and the analogy may also be applied to the present study at lower pH. The reduction of turbidity after heating at 60 and 70 � C may involve mostly disruption of physical bonds such as hydrogen bonding relevant to casein structures (Thomar & Nicolai, 2016), whereas aggregation of whey proteins themselves 3
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Fig. 2. The turbidity and mean hydrodynamic diameter of 5% w/v SMP dispersions after acidification to pH 2.4–3.0 (a) and subsequent heating at 90 � C for 2 min (b), pH 2.4 and heating at 60–90 � C for 10 min (c), or pH 2.4 and heating at 70 (d) or 90 � C (e) for 2–30 min. The unheated pH 6.8 control is shown in Fig. (a) for comparison, with the turbidity beyond the instrument limit of 4000 NTU. Different letters above symbols indicate significant difference in the compared parameter in the same plot (P < 0.05). Error bars are SD (n ¼ 3).
and/or with casein micelles may contribute to observations at 80 and 90 � C (Mulcahy, Fargier-Lagrange, Mulvihill, & O’Mahony, 2017). Polymerization of casein micelles is another mechanism that can in crease turbidity and Dh of dispersions; however, it is an unlikely explanation in this study because heating temperature was lower than 120 � C (El-Din & Aoki, 1993). Structures of dispersions after heating were further examined for morphology detailed below.
corresponding to the right shift of the particle size distribution (Fig. 3e). To understand the relative role of casein and whey protein during heating at pH 2.4, another set of experiments were studied for disper sions with 1.73% w/v MC only and those with 1.39% w/v MC and 0.39% w/v WPI, which has a protein concentration similar to that of 5% w/v SMP dispersions. The significant reductions in the turbidity and Dh of MC dispersions without (Fig. 4, distributions shown in supporting Fig. S2) and with (Fig. 5, distributions shown in supporting Fig. S3) WPI after heating at 70 � C agreed with observations of SMP dispersions (Fig. 2d), confirming the role of casein micelle dissociation, as discussed previously. The results at 90 � C (Figs. 4 and 5, distributions shown in supporting Figs. S2 and S3), however, were opposite to those of SMP dispersions (Fig. 2e). SMP samples have a higher ionic strength than MC samples, and a higher ionic calcium in SMP dispersions favors aggre gation and coagulation of milk proteins during heating due to binding of
3.1.3. Effects of heating duration To further understand effects of heating, turbidity and Dh of dis persions at pH 2.4 were measured after heating at 70 and 90 � C for up to 30 min. Both turbidity and Dh decreased significantly when heating time at 70 � C was extended to 5 min, followed by insignificant changes (Fig. 2d). Conversely, when samples were heated at 90 � C, a longer heating duration increased both turbidity and Dh significantly (Fig. 2e), 4
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Fig. 3. The particle size distributions of 5% w/v SMP dispersions after acidification to pH 2.4–3.0 (a) and subsequent heating at 90 � C for 2 min (b), pH 2.4 and heating at 60–90 � C for 10 min (c), or pH 2.4 and heating at 70 (d) or 90 � C (e) for 2–30 min. The unheated pH 6.8 control is plotted in Fig. (a) for comparison.
calcium ions to carboxylate groups of the protein and subsequently screening charges of caseins, resulting in the increased Dh and turbidity (Gaspard, Auty, Kelly, O’Mahony, & Brodkorb, 2017; On-Nom, Gran dison, & Lewis, 2012). Other studies reported that there was no increase in casein micelle dimension after heating at pH 6.55 when whey protein and lactose were absent (Anema & Li, 2003; Le et al., 2008). The thermal stability of casein micelles is well known and is attributed to κ-casein on micelle surface providing steric repulsion. However, casein micelles are dissociated at the pH range of the present study, but the dissociated caseins can form physical structures to impact the measured parameters. Maillard reaction might occur while lactose and reactive lysine residues
of casein interact; however, it was improbable due to short term of heating, mostly 2 min in the study, at the studied pH range (Crowley et al., 2014). 3.2. Morphological structural changes of dispersions STEM is a valuable tool to characterize nanostructures of materials, where the same principle of scanning electron microscopy (SEM) applies but transmitted electrons can be obtained by scanning a sample after focusing the electron beam (Gee, Carey, & Auty, 2010; Pennycook et al., 2006). Topographical features of SMP dispersions before and after 5
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Fig. 5. Turbidity and mean hydrodynamic diameter of dispersions with 1.39% w/v micellar casein and 0.39% w/v WPI after acidification to pH 2.4 and heating at 70 (a) or 90 � C (b) for 2–30 min. Different letters above symbols indicate significant difference in the compared parameter (P < 0.05). Error bars are SD (n ¼ 3).
Fig. 4. Turbidity and mean hydrodynamic diameter of 1.73% w/v micellar casein dispersions after acidification to pH 2.4 and heating at 70 (a) or 90 � C (b) for 2–30 min. Different letters above symbols indicate significant difference in the compared parameter (P < 0.05). Error bars are SD (n ¼ 3).
acidification and subsequent heating are represented in Fig. 6. Intact casein micelles displayed as darker particles compared to dissociated casein micelles with reduced sphericity. The difference in contrast mainly results from difference in the number of stained particles in the sample (McMahon & Oommen, 2008). Intact casein micelles at pH 6.8 are comprised of a greater number of casein molecules than dissociated casein micelles and accordingly, are stained better to provide higher electron density, resulting in darker structures. The size of intact casein micelles varies from 25 to 300 nm with an average size of 200 nm (de Kruif, 1998). In the present study, this variation was observed from 20 nm to 400 nm at pH 6.8 before heating. The acidified samples had individual casein molecules and loosely associated irregular structures before and after heating, confirming dissociation of casein micelles at pH 2.4–3.0 and explaining multi-modal distribution of particles (Fig. 3b). The decreased dimension of fractal casein structures in acid ified samples after heating agrees with the DLS data (Figs. 2 and 3). When compared to intact casein micelles, some fractal structures of dissociated casein micelles were bigger and had fewer casein molecules (lighter structures in Fig. 6) and thus weaker ability to scatter visible light, which, together with the reduction in particle dimension, con tributes to the reduced dispersion turbidity after acidification and heating (Figs. 1 and 2).
3.3. Structural changes of proteins in skim milk powder dispersions studied with fluorescence spectroscopy The fluorescence spectra resulting from tryptophan (Trp) residues can be obtained at a certain range of excitation wavelength (Teale, 1960). Two Trp residues are present in αs1-casein at 164 and 199 posi tions (Alaimo, Wickham, & Farrell Jr, 1999) and β-casein contains one Trp at 143 position (Kumosinski, Brown, & Farrell Jr, 1993). Trp 164 is located in the hydrophobic domain of casein molecules and is heavily involved in self-association of αs1-casein (Alaimo, Wickham, & Farrell, 1999). The intrinsic fluorescence emission spectra (Fig. 7a) and the wavelength corresponding to the maximum fluorescence intensity (λmax, Table 1) of Trp residues varied among samples. Compared to the pH 6.8 SMP dispersion control, there was a significant increase in emission intensity after acidification to pH 3.0, and the increase in intensity was reduced as dispersions were further acidified to a lower pH of 2.7 and 2.4. At a specific pH, heating at 90 � C for 2 min resulted in a reduction in fluorescence intensity. The λmax shows high sensitivity depending on the exposure of the chromophore. Particularly, a shift of λmax to a longer wavelength (red shift) occurs when there exist positively or negatively charged molecules near the benzene- or pyrrole-ring of Trp (Vivian & Callis, 2001). Compared to samples before heating, a small red shift of about 2 nm was observed in both acidified and neutral SMP dispersions after heating (Table 1). Significant red shifts (~10 nm) can occur when buried Trp residues are exposed to water; however, this is only allowed 6
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Fig. 6. STEM images of 1% w/v SMP dispersions at pH 6.8 (a, b) or acidified to pH 2.4 (c, d), 2.7 (e, f), and 3.0 (g, h), before (a, c, e, g) and after heating (b, d, f, h) at 90 � C for 2 min.
when both benzene and pyrrole rings of Trp residues are exposed to water (Vivian et al., 2001). Thus, the small red shift may be interpreted that heat treatment induced only a minor change in the exposure of Trp residues which agrees with the existence of fractal structures after heating (Fig. 6). The increased exposure of hydrophobic residues in dicates weakened inter-molecular hydrophobic interaction between casein molecules resulting in decreased turbidity of acidified SMP
dispersions after heating (Figs. 1 and 2). As SMP dispersions were acidified from pH 3.0 to 2.4, both λmax and fluorescence intensity decreased (Table 1 and Fig. 7a). This observation may be attributed to two mechanisms: hydrogen bonding and cation-π interaction (Liu et al., 2008). Hydrogen bonds within proteins compete with water. Below the pI of casein, amino acid residues are protonated to a higher extent at a lower pH, and higher affinity of hydrogen ions in 7
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Table 1 The wavelength corresponding to the maximum intrinsic fluorescence (λmax) of 5% w/v SMP dispersions before and after acidification to pH 2.4–3.0, before and after heating at 90 � C for 2 min. pH of SMP dispersion 2.4 2.7 3.0 6.8
λmax (nm) before heating
after heating
348.5 348.5 353 359
350.5 352 355 361.5
can easily interact with highly protonated nitrogen-containing side groups of arginine and lysine (Gallivan & Dougherty, 2000; Liu et al., 2008). The cation-π interaction usually occurs on the surface of proteins, which reduces the exposure of aromatic amino acids to water (Gallivan et al., 2000). These two enhanced interactions after acidification might contribute to the decreased λmax and fluorescence intensity. The low water solubility of pyrene, 0.135 mg/L (Mackay & Shiu, 1977), promotes binding of pyrene with hydrophobic regions of proteins in aqueous solutions (Liu et al., 2008). There are five vibronic band peaks from the fluorescence spectrum of monomeric pyrene in solvents, and the intensity ratio of Band III (~385 nm) to Band I (~375 nm) (I3/I1) provides insights about the polarity of local environment of pyrene (Kalyanasundaram & Thomas, 1977). When surfactant micelles are present, pyrene interacts with hydrophobic domains of aggregates, and the abrupt change in I3/I1 from a pure solvent system can be used as a parameter to determine the critical micellar concentration of surfactants (Kalyanasundaram et al., 1977). I3/I1 of SMP dispersions after acidifi cation and heating did not changed significantly when compared to that of the pH 6.8 control (Table 2), which is different from the expected increase of I3/I1 after acidification due to dissociation of casein micelles. Our results, however, agreed with a previous study (Liu et al., 2008) reporting similar I3/I1 of casein micelle dispersions at extremal pH (e.g., pH 2 and 12) if the concentration of casein was higher than 0.5% (w/v). Therefore, our results confirm that dissociated caseins at a high enough concentration can still associate via hydrophobic amino acid residues to have a similar access by pyrene as the pH 6.8 control. ANS is another fluorescent probe to study protein conformations due to its strong binding affinity to hydrophobic regions of protein (Semi sotnov et al., 1991). When dissolved in water, fluorescence intensity of ANS is quite low, with λmax of ~520 nm; however, binding of ANS to hydrophobic patches of proteins can greatly increase emission intensity while shifting λmax towards a shorter wavelength, i.e., blue shift (Liu et al., 2008; Stryer, 1965). The fluorescence emission spectra of SMP dispersions with ANS are presented in Fig. 7c. The emission intensity of ANS itself is independent on the pH (Liu et al., 2008). Therefore, the increased intensity after acidification of SMP dispersions can be attrib uted to increased binding of ANS to hydrophobic residues of dissociated caseins, which is different from pyrene spectra (Fig. 7b). In addition, ion pairing between the sulfonate group of ANS and the cationic groups such as arginine, lysine, and histidine of proteins (Gasymov & Glasgow, 2007; Matulis & Lovrien, 1998) is enhanced after dissociation of casein mi celles to increase the fluorescence emission intensity. Moreover, the intensity was further increased after heating, which can result from Table 2 I3/I1 of 5% w/v SMP dispersions with 1.0 � 10 6 M pyrene before and after acidification to pH 2.4–3.0, before and after heating at 90 � C for 2 min.
Fig. 7. Fluorescence spectra of 5% w/v SMP dispersions without (a) and with 1.0 � 10 6 M pyrene (b) or 1.0 � 10 5 M ANS (c) before and after acidification to pH 2.4–3.0, before and after heating at 90 � C for 2 min.
pH of SMP dispersion
amino group, –NHþ 3 , than those in –NH2 strengthens hydrogen bond pairing (Liu et al., 2008). In addition, cation-π interaction, i.e., non covalent attraction between cation and electron-rich π system, is favored at acidic pH as aromatic side groups of Trp, the electron-rich π system,
2.4 2.7 3.0 6.8
8
I3/I1 before heating
after heating
1.029 1.009 1.026 1.105
1.029 1.019 1.010 1.124
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further dissociation of casein micelles due to heating (Fig. 2) resulting from weakened hydrophobic interaction between caseins, allowing more ANS binding to exposed hydrophobic patches.
due to weakened hydrophobic interactions rather than further dissolu tion of CCP, which agrees with ANS spectra (Fig. 7C). Increased calcium and phosphorous concentrations in the serum of SMP dispersions were reported after acidification to pH 4.6–6.4 using GDL due to solubiliza tion of calcium from carboxyl groups of glutamate and aspartate or solubilization of phosphorous by release of CCP from micellar phase due to added hydronium (Koutina & Skibsted, 2015). It has been known that some parts of calcium and all the phosphate of CCP are solubilized at pH ~5.2 and calcium is only completely solubilized below pH 3.5, depending on the solubility of specific calcium phosphate complex (Gaucheron, 2005). However, our results suggest that there were still insolubilized calcium and phosphate at pH 3.0, as indicated by continued increases in serum ion concentrations after further acidifi cation (Fig. 8).
3.4. Calcium and phosphorous concentrations in the serum of SMP dispersions Acidification increased (P < 0.05) both calcium and phosphorous concentrations in the serum phase of SMP dispersions, while heating had insignificant effect (P > 0.05) (Fig. 8). The results suggest that calcium and phosphorous were dissolved from CCP in the casein micelles during acidification. A possible mechanism is that charged amino acids in casein molecules, such as negatively charged phosphoserine residues, are neutralized during acidification to cause the diffusion of the attached CCP to the serum (Jaros, Jacob, Otto, & Rohm, 2010). The solubility of CCP itself is also higher at a lower pH while calcium and phosphate ions are stabilized as supersaturated in equilibrium with CCP at neutral pH. Additionally, citrate ions (pKa1 ¼ 3.13, pKa2 ¼ 4.76, and pKa3 ¼ 6.40) are known metal chelators (Meier-Kriesche, Finkel, Gitomer, & DuBose, 1999). Because CCP is critical to the structure of casein micelles, results in Fig. 8 explain the decreased Dh after acidification and subsequent heating (Fig. 2) resulted from dissociation of casein micelles by the above mentioned factors to result in the reduced sample turbidity. The insignificant changes in concentrations of calcium and phosphorous after heating suggest that reductions in turbidity after heating may be
3.5. Effects of acidification and heating on free thiol concentration and molecular weight of proteins Comparison of molecular weight between acidified samples before and after heating was analyzed using polyacrylamide gel electrophoresis
Fig. 8. Calcium (a) and phosphorous (b) concentrations in the serum phase of 5% w/v SMP dispersions (5% w/v) before and after acidification to pH 2.4–3.0, before and after heating at 90 � C for 2 min. Different letters above bars indicate significant difference in the compared parameter (P < 0.05). Error bars are SD (n ¼ 3).
Fig. 9. Reducing (a,b) and non-reducing (c,d) SDS-PAGE of 5% w/v SMP dis persions before (pH 6.8, far right lane in each panel of four lanes) and after acidification to pH 2.4, 2.7, and 3.0 (left to right in each panel of four lanes) before (a,c) and after (b,d) heating at 90 � C for 2 min. 9
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(PAGE). As shown in Fig. 9(a–d), the intensity of each band was similar irrespective of pH and heating in both reducing and non-reducing PAGE. This is similar to another reducing SDS-PAGE study (Ranadheera et al., 2019) showing no apparent change in band intensity of milk protein concentrate dispersions between pH 2 and pH 4.6. Similar band in tensity and pattern of SMP dispersions suggest either little inter-molecular disulfide bonds formed after acidification and heating or the changes were not detectable at the studied conditions. Acidification from pH 6.8 to 2.4 before heating decreased the amount of free thiol groups (Fig. 10). At pH 6.8, β-Lg exists as dimers and is arranged as open conformation; however, lowering pH causes closed conformation of β-Lg by moving E-F loop (residues 86–89), resulting in less access to amino acid residues, i.e., free thiol groups (Uhrínov� a et al., 2000). The concentration of free thiol groups decreased significantly (P < 0.05) after heating samples at pH 6.8. Disulfide bonding due to sulfhydryl-disulfide exchange is the major mechanism governing the formation of whey protein-casein micelle complexes at pH 6.7 (Jang et al., 1990). This suggests that the decreased number of free thiol groups involves with disulfide bonding between whey proteins, partic ularly β-Lg, or between whey proteins and caseins, e.g., β-Lg and κ-casein after the heating at pH 6.8. At pH 3, the concentration of free thiol group did not change significantly after heating, which is in agreement with a previous study (Monahan, German, & Kinsella, 1995). No reduction in free thiol group content at acidic pH is expected, as formation of disulfide bonds is inhibited due to limited reactivity of free thiol groups at acidic pH (Alting, de Jongh, Visschers, & Simons, 2002). However, heating resulted in the increased free thiol groups at pH 2.4 and 2.7, possibly due to further dissociation of casein micelles causing the increased exposure of free thiol groups. Combining all the results above, however, there is still no clear evidence resulting a vivid band emerged at ~80 kDa on the reducing SDS-PAGE (Fig. 9a and b).
Fig. 10. Contents of free thiol groups in the 5% w/v SMP dispersions before and after acidification to pH 2.4–3.0, before and after subsequent heating at 90 � C for 2 min. Different letters above bars indicate significant difference (P < 0.05). Error bars are SD (n ¼ 3).
Declaration of competing interest We do not have any undisclosed relationship and funding source that may pose a competing interest. Acknowledgement The authors are grateful to Dr. John Dunlap, Dr. Michael Essington, Dr. Edward Wright, and Melanie Stewart for assisting with experiments. This study was supported by USDA National Institute of Food and Agriculture, TEN2015-05921 and hatch projects TEN00487 and 223984. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture.
4. Conclusion Acidification of SMP dispersion to pH 3.0 with citric acid increased the solubility of CCP and, together with calcium-chelating properties of citrate resulted in dissociation of casein micelles and reduction of dispersion turbidity. The dissociated caseins formed open fractal struc tures at pH 3.0, involving hydrophobic interactions, hydrogen bonds and cation-π interactions according to fluorescence spectroscopy, to result in the higher Dh than the SMP dispersion at pH 6.8, but the low refractive index of fractal structures was responsible for the lowered dispersion turbidity at pH 3.0. Upon further acidification to pH 2.7 and 2.4, the continued dissolution of CCP further reduced the dimension of casein micelles and, together with the reduced refractive index, resulted in the lower turbidity and smaller Dh than the SMP dispersion at pH 6.8. Upon heating at pH 2.4, no disulfide bond formation, based on SDSPAGE and free thiol content, and no significant changes in serum cal cium and phosphate concentrations were detected at the studied con ditions, and hydrophobic interactions between caseins were weakened to reduce the dimension of fractal structures to further reduce SMP dispersion turbidity and Dh. With extended heating at pH 2.4, whey protein denaturation was insignificant at 70 � C and the SMP dispersion showed insignificant changes in turbidity and Dh. However, whey pro tein denaturation was possible at 90 � C, and the calcium ions in the serum and the increased ionic strength relative to MC and MC/WPI dispersions may have facilitated the aggregation of proteins in SMP dispersions to cause the continued increase in Dh at a longer heating time. The open fractal structures with a low refractive index were also responsible for insignificant changes in SMP dispersion turbidity after extended heating at 90 � C. The exact mechanism of serum ions impacting structures of SMP dispersions however is to be further stud ied. Nevertheless, findings from the present study may be used to guide the incorporation of SMP in relevant translucent protein-fortified beverages.
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Physicochemical properties of skim milk powder dispersions after acidification to pH 2.4–3.0 and heating Inseob Choi, Qixin Zhong * Department of Food Science, The University of Tennessee, Knoxville, TN, 37996, United States
A R T I C L E I N F O
A B S T R A C T
Keywords: Skim milk powder dispersion Acidification Heating Casein micelle Colloidal calcium phosphate Hydrophobic interaction
Application of skim milk powder (SMP) in acidified beverages is limited due to precipitation and sedimentation of caseins. In this study, 5% w/v SMP dispersion was acidified to pH 2.4–3.0 using citric acid and heated at different conditions. Lowering pH resulted in dissolution of colloidal calcium phosphate from casein micelles, corresponding to lowered turbidity and reduced hydrodynamic diameter of dispersions. When heated for 10 min at different temperatures, 70 � C was the most effective to produce transparent dispersions; whereas a shorter heating time between 2 and 60 min was desired at 90 � C. The decreased turbidity after heating was attributed to weakened hydrophobic interaction between casein molecules based on fluorescence spectrometry and ion analysis. Scanning transmission electron microscopy showed the altered structure and decreased dimension of casein micelles after acidification and heating. The current study may expand the use of SMP for manufacturing new types of protein drinks.
1. Introduction Skim milk powder (SMP) contains high-quality proteins and a fat content of less than 1.2% wt (Devine, Bell, & Prince, 1996). Due to its long shelf-life and high protein content, SMP has been used in a wide range of applications including reconstituted and recombined dairy products (Anema & McKenna, 1996), confectionery (Liang & Hartel, €hko €nen, Tuorila, & Hyvo €nen, 1995), and diet beverages 2004), soups (Ka (Mesirow & Welsh, 2015). Casein micelles constitute 80% of milk pro teins and play an important role to determine physicochemical proper ties of SMP dispersions; the other 20% milk proteins are whey proteins. Despite nutritional quality of SMP, the precipitation of casein micelles limits the use of SMP in acidic beverages without hydrocolloid stabi lizers. Additionally, beverages with lowered turbidity are perceived by consumers as healthier, natural, and less contaminated products (Tse & Yim, 2002). Studies are therefore needed to dissociate casein micelles to reduce turbidity of and stabilize caseins in acidified beverages. The structure of casein micelles is enabled by a balance between attractive hydrophobic and repulsive electrostatic interactions, as well as colloidal calcium phosphate (CCP) bridging calcium sensitive ɑs1-and β-caseins that are highly phosphorylated (Liu & Guo, 2008). The CCP bridging strengthens the short-range hydrophobic interactions between hydrophobic regions of caseins and neutralizes negatively charged
phosphoserine groups (Liu et al., 2008). The knowledge of interactions in casein micelles can be used to guide studies to reduce milk turbidity. CCP is in equilibrium with free calcium and phosphate ions in the serum phase of milk (Walstra, 1999), and chelating agents with a stronger binding affinity to calcium than phosphorylated caseins result in the dissolving of CCP and therefore dissociation of casein micelles (Kaliap pan & Lucey, 2011; Lazzaro et al., 2017). The dissociation of casein micelles has also been reported after high-pressure treatments that disrupt CCP linkages with casein (Bravo, Felipe, Lopez-Fandino, & Molina, 2013). Acidification from pH 6.0 to pH 4.0 results in the increased concentrations of calcium and phosphate in milk serum, suggesting the dissolving of CCP in casein micelles (Le Gra€et & Gau cheron, 1999); acidification however weakens electrostatic repulsion to cause aggregation of caseins. Thermal treatment is used to ensure the safety and shelf-life of milk. When skim milk is heated, heterogeneous casein-whey protein com plexes can be formed with whey proteins depositing onto casein micelles (Dalgleish, van Mourik, & Corredig, 1997). Heating milk at 70–90 � C causes the denaturation and exposure of the buried reactive thiol groups of beta-lactoglobulin (β-Lg) (Le et al., 2007) to form disulfide bonds between β-Lg and κ-casein on casein micelle surface by thiol-disulfide interchange reactions (Jang & Swaisgood, 1990). This has been shown for the maintained particle size distribution of milk depleted for whey
* Corresponding author. E-mail address: [email protected] (Q. Zhong). https://doi.org/10.1016/j.foodhyd.2019.105435 Received 24 July 2019; Received in revised form 10 October 2019; Accepted 10 October 2019 Available online 14 October 2019 0268-005X/© 2019 Elsevier Ltd. All rights reserved.
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proteins after heating (Le, Saveyn, Hoa, & Van der Meeren, 2008). The extent of association between whey proteins and casein micelles is greater at a lower pH in the range from 7.3 to 5.8 (Smits & van Brou wershaven, 1980). Despite popularity in Europe and Asia, acidified milk products are susceptible to sedimentation due to casein aggregation around pH 3.8–5.0 (Amice-Quemeneur, Haluk, Hardy, & Kravtchenko, 1995). Regardless of differences in the composition of casein micelles and mineral balance in skim milk, gelation occurs when milk is subjected to acidification (e.g., ~pH 4.2) using glucono-δ-lactone (GDL) (Ould Eleya, Desobry Banon, & Hardy, 1995). Additionally, the traditional acidifi cation method using GDL has poor reproducibility (Amice-Quemeneur et al., 1995). Physicochemical properties of skim milk and casein mi celles below pH 3.8 have not been reported but are significant to many acidic beverages that can be potentially fortified with casein-based dairy proteins. The overall objective of this study was to reduce the turbidity of SMP dispersions at pH 2.4–3.0 and characterize physicochemical properties of SMP dispersions after acidification with citric acid and heating. Citric acid was used as it is commonly used in food beverage production and citrate is a well-known chelating agent. The fundamental information was studied to understand turbidity changes of SMP dispersions using dynamic light scattering (DLS), fluorescence spectrometry, inductively coupled argon plasma-optical emission spectrometry (ICP-OES), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), freethiol (SH) group concentrations, and scanning transmission electron microscopy (STEM).
used to measure the particle size distribution of dispersions (model Zetasizer nano-ZS, Malvern Instrument, Worcestershire, UK). The mean hydrodynamic diameter (Dh) was calculated using Zetasizer Software 7.11. Measurements were done with a backscatter angle of 173� and an equilibrium temperature of 25 � C. 2.4. Scanning transmission electron microscopy The morphology of particulates before and after acidification and after heating at 90 � C for 2 min was studied using STEM. The 5% w/v SMP dispersions were diluted five times with the corresponding serum obtained using the centrifugal ultrafiltration method detailed in the below ICP-OES section. The diluted 1% w/v SMP dispersion was placed on a piece of Parafilm. A freshly glow discharged 400 mesh copper grid with a thin carbon film was placed on the sample drop. After 1 min, excess sample was quickly removed from the grid and the grid was placed on a drop of water for 10 s. The water was removed using Kim wipes™ and the grid was placed on a drop of 1% uranyl acetate for 1 min. Then excess stain was quickly removed using Kimwipes™ and the sample was allowed to dry at RT. The stained samples were imaged using a Zeiss Auriga microscope (Carl Zeiss, Thornwood, NY) operating in the STEM mode at 30 kV. 2.5. Fluorescence spectrometry Steady state fluorescence spectra were measured using a LS 55 fluorescence spectrometer (PerkinElmer, Waltham, MA) to study effects of acidification and heating on the structure of proteins in SMP disper sions. To acquire intrinsic fluorescence spectra, the excitation wave length was 295 nm and the range of emission spectra was between 300 and 500 nm. Pyrene and 8-anilinonaphthalene-1-sulfonic acid (ANS) were used individually at 1.0 � 10 6 and 1.0 � 10 5 M, respectively, in 5% w/v SMP dispersions, as probes binding to hydrophobic sites of proteins, and the spectra were obtained at an excitation wavelength of 338 and 380 nm, respectively. The emission spectra were collected from 350 to 600 nm for SMP dispersions with pyrene and from 400 to 560 nm for those with ANS (Liu et al., 2008). Excitation and emission slit sizes were fixed at 5.0 nm for intrinsic fluorescence spectra and pyrene treatments and 2.5 nm for ANS treatments. The scan rate for all mea surements was set as 240 nm/min.
2. Materials and methods 2.1. Materials Carnation® SMP (Nestle� USA, Solon, OH) was used in the present study. The SMP was determined to contain 35.4% protein based on the Kjeldahl method (AOAC, 2005). The calcium content in SMP was determined to be 2.10% after dry ashing and ICP-OES detailed below. Citric acid was purchased from Fisher Scientific (Fair Lawn, NJ). Micellar casein (MC) was from American Casein Company (Burlington, NJ) and whey protein isolate (WPI) was from Hilmar Ingredients (Hil mar, CA). Glycine and Ellman’s reagent were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Tris, ethylenediaminetetraacetic acid, and urea were procured from Acros Organics (Pittsburgh, PA).
2.6. Inductively coupled plasma-optical emission spectrometry
2.2. Sample preparation
To investigate the effect of acidification and heating on the disso lution of CCP, acidified SMP dispersions were determined for serum calcium and phosphorous concentrations before and after heating at 90 � C for 2 min. The Amicon ultra-15 centrifugal filter unit having an ultracel-10 membrane with a molecular weight cut-off of 10,000 Da (Merck Millipore Ltd, Cork, Ireland) was used to obtain the serum phase of SMP dispersions. Centrifugation was performed at 4500 g for 3 h using Sorvall RC-5B plus centrifuge (Sorvall, Newtown, CT). Each serum was diluted 100 times with deionized water and analyzed using a Spectro Ciros ICP-OES instrument (Spectro Ciros, Mahwah, NJ). To calibrate the instrument before calcium and phosphorous analysis, ICP-OES standards were used (VHG Labs, Manchester, NH). The detection limit for calcium and phosphorous in the method was both 0.01 mg/L.
SMP was hydrated with deionized water at 5% w/v in 20 mL scin tillation vials. After vortexing, samples were incubated at room tem perature (RT, ~21 � C) for at least 8 h for complete hydration. Citric acid solution (2.0 M) was added drop-wise in the SMP dispersions until reaching pH 2.4, 2.7, and 3.0. The SMP dispersion at pH 6.8 without acidification was used as a control. To study interactions between casein micelles and whey proteins, dispersions with 1.73% w/v MC or 1.39% w/v MC and 0.39% w/v WPI, simulating the proximate mass ratio of casein and whey protein (4:1) in bovine milk (Zadow, 1994), were similarly prepared. Vials were incubated in a water bath pre-equilibrated to 60, 70, 80, and 90 � C for 2, 5, 10, 30, and 60 min. Thermal profiles of sample centers are shown in supporting Fig. S1 to guide future scale-up attempts. As the thermal come-up time varied, the treatments are still named at the set temperature for convenience in description.
2.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SMP dispersions without or with heating at 90 � C for 2 min were analyzed for both reducing and non-reducing SDS-PAGE. The sample buffer was prepared with β-mercaptoethanol and Laemmli sample buffer at a volume ratio of 5:95 for reducing SDS-PAGE, and β-mercaptoethanol was excluded for non-reducing SDS-PAGE. Each SMP dispersion was diluted 40 times with the sample buffer. Samples were heated in boiling water for 5 min before reducing SDS-PAGE. The running buffer for gel
2.3. Turbidity and particle size distribution Turbidity of SMP dispersions was measured at RT using a 2020 we/ wi turbidimeter (LaMotte, Chestertown, MD). Calibration was per formed using the AMCO primary turbidity standard (10 NTU). DLS was 2
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electrophoresis was prepared with 100 mL of 10� Tris/Glycine/SDS and 900 mL of deionized water. Ten microliters of a sample were loaded in each well of 4–20% Mini-PROTEAN TGX Gels (Bio-Rad Laboratories, Hercules, CA). The electrophoresis was run at 300 mA and 150 V for 2 h. After washing with deionized water three times for 5 min, the gels were stained using Bio-safe Coomassie G-250 Stain for 1 h and then rinsed with deionized water for 30 min. 2.8. Determination of free thiol (SH) group concentration Free thiol (SH) groups in SMP dispersions before and after acidifi cation and heating were determined using the method of Beveridge et al. (Beveridge, Toma, & Nakai, 1974) with slight modification. SMP dis persions prepared as above at pH 2.4, 2.7, or 3.0 were heated at 90 � C for 2 min. Two hundred microliters of a SMP dispersion were mixed in 1 mL of Tris-glycine buffer containing 0.086 M Tris, 0.09 M glycine, 4 mM ethylenediaminetetraacetic acid, and 8 M urea, and reacted with 8 μL of Ellman’s reagent containing 10 mM 5,50 -dithiobis (2-nitrobenzoic acid). After centrifugation at 10,000 g for 5 min (model 4540, Eppendorf, Hamburg, Germany), absorbance of supernatant was measured at 412 nm using a model Evolution 201 UV–Vis spectrophotometer (Thermo Scientific, Waltham, MA). The concentration of SH groups was determined using Beer’s law with the molar extinction coefficient of 13, 600 M 1cm 1. 2.9. Statistical analysis Three replicates of each sample on each test were prepared for sta tistical analysis. One-way analysis of variance (ANOVA) was performed using SAS enterprise guide 7.1 (SAS Institute Inc., Cary, NC). Mean and standard deviation (SD) were calculated and differences of calculated means were considered significant with P < 0.05 using Fisher’s least significance difference (LSD) test. 3. Results and discussion 3.1. Turbidity, hydrodynamic diameter, and particle size distribution of skim milk powder dispersions
Fig. 1. The appearance of 5% w/v SMP dispersions after acidification to pH 2.4–3.0 (a) and heating at 90 � C for 2 min (b), pH 2.4 and heating at 60–90 � C for 10 min (c), and pH 2.4 and heating at 90 � C for 2–30 min (d). The far-left sample is the unheated control at pH 6.8.
The appearance, turbidity and mean Dh, and particle size distribution of skim milk powder dispersions after various treatments are presented in Figs. 1–3, respectively. The results are presented according to the treatment variable.
study was to reduce SMP dispersion turbidity, the rest of heating treatments were studied at pH 2.4.
3.1.1. Effects of acidification Visual appearance of protein-containing beverages is an important quality parameter and is determined by the dimension, concentration, light refraction properties (refractive index), and stability of protein structures (Wagoner & Foegeding, 2017; Zhang & Reineccius, 2016). After acidification to a lower pH, SMP dispersions became visually more translucent (Fig. 1a). Correspondingly, the turbidity and Dh were smaller at a lower pH (Fig. 2a). All acidified dispersions showed a greater portion of particles smaller than the control, and the extent of casein micelle dissociation was greater at a lower pH (Fig. 3a). The less turbid appearance (Fig. 1a) but bigger Dh (Fig. 2a) of the pH 3.0 sample than the control at pH 6.8 suggest the aggregation of initially dissociated caseins. The results are in agreement with the observed casein micelle dissociation at pH 4.0 (Le Gra€ et et al., 1999). The re-aggregated casein particles may have a lower refractive index than casein micelles that are cross-linked by CCP, which causes less light scattering and a lower turbidity at pH 3.0 than at pH 6.8 (Griffin & Griffin, 1985). After heating at 90 � C for 2 min, similar to thermal treatment conditions (88–96 � C for 2 min) in the hot-fill process used to pasteurize high-acid beverages, all dispersions showed the decreases in turbidity and Dh (Fig. 2a vs. 2b) and the narrowed particle size distribution (Fig. 3b), and precipitation was observed for the pH 3.0 dispersion (Fig. 1b). Since the goal of the present
3.1.2. Effects of heating temperature When the pH 2.4 sample was heated for 10 min at 60–90 � C, turbidity of all samples decreased after heating (Fig. 2c vs. 2a), even in the sample heated at 60 � C which was more turbid than other samples (Figs. 1c and 2c). Heating dispersions at 70 � C resulted in the lowest turbidity and the smallest Dh (Fig. 2c). The particle size distributions were more mono dispersed after heating at 80 and 90 � C than at 60 and 70 � C (Fig. 3c), and there was no linear correlation between heating temperature and turbidity or Dh. The results in Fig. 2c suggests two different phenomena below and above 70 � C. The reported denaturation temperature of β-Lg is 75.9, 81.9, and 78.7 � C at pH 6.5, 3.5, and 2.5, respectively, while the respective denaturation temperature of whey protein mixtures at pH 6.5, 3.5, and 2.5 is 76.9, 88.0, and 80.6 � C (Bernal & Jelen, 1985). Above 70 � C, a positive relationship was reported between temperature and the amount of denatured whey proteins, leading to increases in turbidity and Dh of milk (Dannenberg & Kessler, 1988), and the analogy may also be applied to the present study at lower pH. The reduction of turbidity after heating at 60 and 70 � C may involve mostly disruption of physical bonds such as hydrogen bonding relevant to casein structures (Thomar & Nicolai, 2016), whereas aggregation of whey proteins themselves 3
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Fig. 2. The turbidity and mean hydrodynamic diameter of 5% w/v SMP dispersions after acidification to pH 2.4–3.0 (a) and subsequent heating at 90 � C for 2 min (b), pH 2.4 and heating at 60–90 � C for 10 min (c), or pH 2.4 and heating at 70 (d) or 90 � C (e) for 2–30 min. The unheated pH 6.8 control is shown in Fig. (a) for comparison, with the turbidity beyond the instrument limit of 4000 NTU. Different letters above symbols indicate significant difference in the compared parameter in the same plot (P < 0.05). Error bars are SD (n ¼ 3).
and/or with casein micelles may contribute to observations at 80 and 90 � C (Mulcahy, Fargier-Lagrange, Mulvihill, & O’Mahony, 2017). Polymerization of casein micelles is another mechanism that can in crease turbidity and Dh of dispersions; however, it is an unlikely explanation in this study because heating temperature was lower than 120 � C (El-Din & Aoki, 1993). Structures of dispersions after heating were further examined for morphology detailed below.
corresponding to the right shift of the particle size distribution (Fig. 3e). To understand the relative role of casein and whey protein during heating at pH 2.4, another set of experiments were studied for disper sions with 1.73% w/v MC only and those with 1.39% w/v MC and 0.39% w/v WPI, which has a protein concentration similar to that of 5% w/v SMP dispersions. The significant reductions in the turbidity and Dh of MC dispersions without (Fig. 4, distributions shown in supporting Fig. S2) and with (Fig. 5, distributions shown in supporting Fig. S3) WPI after heating at 70 � C agreed with observations of SMP dispersions (Fig. 2d), confirming the role of casein micelle dissociation, as discussed previously. The results at 90 � C (Figs. 4 and 5, distributions shown in supporting Figs. S2 and S3), however, were opposite to those of SMP dispersions (Fig. 2e). SMP samples have a higher ionic strength than MC samples, and a higher ionic calcium in SMP dispersions favors aggre gation and coagulation of milk proteins during heating due to binding of
3.1.3. Effects of heating duration To further understand effects of heating, turbidity and Dh of dis persions at pH 2.4 were measured after heating at 70 and 90 � C for up to 30 min. Both turbidity and Dh decreased significantly when heating time at 70 � C was extended to 5 min, followed by insignificant changes (Fig. 2d). Conversely, when samples were heated at 90 � C, a longer heating duration increased both turbidity and Dh significantly (Fig. 2e), 4
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Fig. 3. The particle size distributions of 5% w/v SMP dispersions after acidification to pH 2.4–3.0 (a) and subsequent heating at 90 � C for 2 min (b), pH 2.4 and heating at 60–90 � C for 10 min (c), or pH 2.4 and heating at 70 (d) or 90 � C (e) for 2–30 min. The unheated pH 6.8 control is plotted in Fig. (a) for comparison.
calcium ions to carboxylate groups of the protein and subsequently screening charges of caseins, resulting in the increased Dh and turbidity (Gaspard, Auty, Kelly, O’Mahony, & Brodkorb, 2017; On-Nom, Gran dison, & Lewis, 2012). Other studies reported that there was no increase in casein micelle dimension after heating at pH 6.55 when whey protein and lactose were absent (Anema & Li, 2003; Le et al., 2008). The thermal stability of casein micelles is well known and is attributed to κ-casein on micelle surface providing steric repulsion. However, casein micelles are dissociated at the pH range of the present study, but the dissociated caseins can form physical structures to impact the measured parameters. Maillard reaction might occur while lactose and reactive lysine residues
of casein interact; however, it was improbable due to short term of heating, mostly 2 min in the study, at the studied pH range (Crowley et al., 2014). 3.2. Morphological structural changes of dispersions STEM is a valuable tool to characterize nanostructures of materials, where the same principle of scanning electron microscopy (SEM) applies but transmitted electrons can be obtained by scanning a sample after focusing the electron beam (Gee, Carey, & Auty, 2010; Pennycook et al., 2006). Topographical features of SMP dispersions before and after 5
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Fig. 5. Turbidity and mean hydrodynamic diameter of dispersions with 1.39% w/v micellar casein and 0.39% w/v WPI after acidification to pH 2.4 and heating at 70 (a) or 90 � C (b) for 2–30 min. Different letters above symbols indicate significant difference in the compared parameter (P < 0.05). Error bars are SD (n ¼ 3).
Fig. 4. Turbidity and mean hydrodynamic diameter of 1.73% w/v micellar casein dispersions after acidification to pH 2.4 and heating at 70 (a) or 90 � C (b) for 2–30 min. Different letters above symbols indicate significant difference in the compared parameter (P < 0.05). Error bars are SD (n ¼ 3).
acidification and subsequent heating are represented in Fig. 6. Intact casein micelles displayed as darker particles compared to dissociated casein micelles with reduced sphericity. The difference in contrast mainly results from difference in the number of stained particles in the sample (McMahon & Oommen, 2008). Intact casein micelles at pH 6.8 are comprised of a greater number of casein molecules than dissociated casein micelles and accordingly, are stained better to provide higher electron density, resulting in darker structures. The size of intact casein micelles varies from 25 to 300 nm with an average size of 200 nm (de Kruif, 1998). In the present study, this variation was observed from 20 nm to 400 nm at pH 6.8 before heating. The acidified samples had individual casein molecules and loosely associated irregular structures before and after heating, confirming dissociation of casein micelles at pH 2.4–3.0 and explaining multi-modal distribution of particles (Fig. 3b). The decreased dimension of fractal casein structures in acid ified samples after heating agrees with the DLS data (Figs. 2 and 3). When compared to intact casein micelles, some fractal structures of dissociated casein micelles were bigger and had fewer casein molecules (lighter structures in Fig. 6) and thus weaker ability to scatter visible light, which, together with the reduction in particle dimension, con tributes to the reduced dispersion turbidity after acidification and heating (Figs. 1 and 2).
3.3. Structural changes of proteins in skim milk powder dispersions studied with fluorescence spectroscopy The fluorescence spectra resulting from tryptophan (Trp) residues can be obtained at a certain range of excitation wavelength (Teale, 1960). Two Trp residues are present in αs1-casein at 164 and 199 posi tions (Alaimo, Wickham, & Farrell Jr, 1999) and β-casein contains one Trp at 143 position (Kumosinski, Brown, & Farrell Jr, 1993). Trp 164 is located in the hydrophobic domain of casein molecules and is heavily involved in self-association of αs1-casein (Alaimo, Wickham, & Farrell, 1999). The intrinsic fluorescence emission spectra (Fig. 7a) and the wavelength corresponding to the maximum fluorescence intensity (λmax, Table 1) of Trp residues varied among samples. Compared to the pH 6.8 SMP dispersion control, there was a significant increase in emission intensity after acidification to pH 3.0, and the increase in intensity was reduced as dispersions were further acidified to a lower pH of 2.7 and 2.4. At a specific pH, heating at 90 � C for 2 min resulted in a reduction in fluorescence intensity. The λmax shows high sensitivity depending on the exposure of the chromophore. Particularly, a shift of λmax to a longer wavelength (red shift) occurs when there exist positively or negatively charged molecules near the benzene- or pyrrole-ring of Trp (Vivian & Callis, 2001). Compared to samples before heating, a small red shift of about 2 nm was observed in both acidified and neutral SMP dispersions after heating (Table 1). Significant red shifts (~10 nm) can occur when buried Trp residues are exposed to water; however, this is only allowed 6
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Fig. 6. STEM images of 1% w/v SMP dispersions at pH 6.8 (a, b) or acidified to pH 2.4 (c, d), 2.7 (e, f), and 3.0 (g, h), before (a, c, e, g) and after heating (b, d, f, h) at 90 � C for 2 min.
when both benzene and pyrrole rings of Trp residues are exposed to water (Vivian et al., 2001). Thus, the small red shift may be interpreted that heat treatment induced only a minor change in the exposure of Trp residues which agrees with the existence of fractal structures after heating (Fig. 6). The increased exposure of hydrophobic residues in dicates weakened inter-molecular hydrophobic interaction between casein molecules resulting in decreased turbidity of acidified SMP
dispersions after heating (Figs. 1 and 2). As SMP dispersions were acidified from pH 3.0 to 2.4, both λmax and fluorescence intensity decreased (Table 1 and Fig. 7a). This observation may be attributed to two mechanisms: hydrogen bonding and cation-π interaction (Liu et al., 2008). Hydrogen bonds within proteins compete with water. Below the pI of casein, amino acid residues are protonated to a higher extent at a lower pH, and higher affinity of hydrogen ions in 7
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Table 1 The wavelength corresponding to the maximum intrinsic fluorescence (λmax) of 5% w/v SMP dispersions before and after acidification to pH 2.4–3.0, before and after heating at 90 � C for 2 min. pH of SMP dispersion 2.4 2.7 3.0 6.8
λmax (nm) before heating
after heating
348.5 348.5 353 359
350.5 352 355 361.5
can easily interact with highly protonated nitrogen-containing side groups of arginine and lysine (Gallivan & Dougherty, 2000; Liu et al., 2008). The cation-π interaction usually occurs on the surface of proteins, which reduces the exposure of aromatic amino acids to water (Gallivan et al., 2000). These two enhanced interactions after acidification might contribute to the decreased λmax and fluorescence intensity. The low water solubility of pyrene, 0.135 mg/L (Mackay & Shiu, 1977), promotes binding of pyrene with hydrophobic regions of proteins in aqueous solutions (Liu et al., 2008). There are five vibronic band peaks from the fluorescence spectrum of monomeric pyrene in solvents, and the intensity ratio of Band III (~385 nm) to Band I (~375 nm) (I3/I1) provides insights about the polarity of local environment of pyrene (Kalyanasundaram & Thomas, 1977). When surfactant micelles are present, pyrene interacts with hydrophobic domains of aggregates, and the abrupt change in I3/I1 from a pure solvent system can be used as a parameter to determine the critical micellar concentration of surfactants (Kalyanasundaram et al., 1977). I3/I1 of SMP dispersions after acidifi cation and heating did not changed significantly when compared to that of the pH 6.8 control (Table 2), which is different from the expected increase of I3/I1 after acidification due to dissociation of casein micelles. Our results, however, agreed with a previous study (Liu et al., 2008) reporting similar I3/I1 of casein micelle dispersions at extremal pH (e.g., pH 2 and 12) if the concentration of casein was higher than 0.5% (w/v). Therefore, our results confirm that dissociated caseins at a high enough concentration can still associate via hydrophobic amino acid residues to have a similar access by pyrene as the pH 6.8 control. ANS is another fluorescent probe to study protein conformations due to its strong binding affinity to hydrophobic regions of protein (Semi sotnov et al., 1991). When dissolved in water, fluorescence intensity of ANS is quite low, with λmax of ~520 nm; however, binding of ANS to hydrophobic patches of proteins can greatly increase emission intensity while shifting λmax towards a shorter wavelength, i.e., blue shift (Liu et al., 2008; Stryer, 1965). The fluorescence emission spectra of SMP dispersions with ANS are presented in Fig. 7c. The emission intensity of ANS itself is independent on the pH (Liu et al., 2008). Therefore, the increased intensity after acidification of SMP dispersions can be attrib uted to increased binding of ANS to hydrophobic residues of dissociated caseins, which is different from pyrene spectra (Fig. 7b). In addition, ion pairing between the sulfonate group of ANS and the cationic groups such as arginine, lysine, and histidine of proteins (Gasymov & Glasgow, 2007; Matulis & Lovrien, 1998) is enhanced after dissociation of casein mi celles to increase the fluorescence emission intensity. Moreover, the intensity was further increased after heating, which can result from Table 2 I3/I1 of 5% w/v SMP dispersions with 1.0 � 10 6 M pyrene before and after acidification to pH 2.4–3.0, before and after heating at 90 � C for 2 min.
Fig. 7. Fluorescence spectra of 5% w/v SMP dispersions without (a) and with 1.0 � 10 6 M pyrene (b) or 1.0 � 10 5 M ANS (c) before and after acidification to pH 2.4–3.0, before and after heating at 90 � C for 2 min.
pH of SMP dispersion
amino group, –NHþ 3 , than those in –NH2 strengthens hydrogen bond pairing (Liu et al., 2008). In addition, cation-π interaction, i.e., non covalent attraction between cation and electron-rich π system, is favored at acidic pH as aromatic side groups of Trp, the electron-rich π system,
2.4 2.7 3.0 6.8
8
I3/I1 before heating
after heating
1.029 1.009 1.026 1.105
1.029 1.019 1.010 1.124
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further dissociation of casein micelles due to heating (Fig. 2) resulting from weakened hydrophobic interaction between caseins, allowing more ANS binding to exposed hydrophobic patches.
due to weakened hydrophobic interactions rather than further dissolu tion of CCP, which agrees with ANS spectra (Fig. 7C). Increased calcium and phosphorous concentrations in the serum of SMP dispersions were reported after acidification to pH 4.6–6.4 using GDL due to solubiliza tion of calcium from carboxyl groups of glutamate and aspartate or solubilization of phosphorous by release of CCP from micellar phase due to added hydronium (Koutina & Skibsted, 2015). It has been known that some parts of calcium and all the phosphate of CCP are solubilized at pH ~5.2 and calcium is only completely solubilized below pH 3.5, depending on the solubility of specific calcium phosphate complex (Gaucheron, 2005). However, our results suggest that there were still insolubilized calcium and phosphate at pH 3.0, as indicated by continued increases in serum ion concentrations after further acidifi cation (Fig. 8).
3.4. Calcium and phosphorous concentrations in the serum of SMP dispersions Acidification increased (P < 0.05) both calcium and phosphorous concentrations in the serum phase of SMP dispersions, while heating had insignificant effect (P > 0.05) (Fig. 8). The results suggest that calcium and phosphorous were dissolved from CCP in the casein micelles during acidification. A possible mechanism is that charged amino acids in casein molecules, such as negatively charged phosphoserine residues, are neutralized during acidification to cause the diffusion of the attached CCP to the serum (Jaros, Jacob, Otto, & Rohm, 2010). The solubility of CCP itself is also higher at a lower pH while calcium and phosphate ions are stabilized as supersaturated in equilibrium with CCP at neutral pH. Additionally, citrate ions (pKa1 ¼ 3.13, pKa2 ¼ 4.76, and pKa3 ¼ 6.40) are known metal chelators (Meier-Kriesche, Finkel, Gitomer, & DuBose, 1999). Because CCP is critical to the structure of casein micelles, results in Fig. 8 explain the decreased Dh after acidification and subsequent heating (Fig. 2) resulted from dissociation of casein micelles by the above mentioned factors to result in the reduced sample turbidity. The insignificant changes in concentrations of calcium and phosphorous after heating suggest that reductions in turbidity after heating may be
3.5. Effects of acidification and heating on free thiol concentration and molecular weight of proteins Comparison of molecular weight between acidified samples before and after heating was analyzed using polyacrylamide gel electrophoresis
Fig. 8. Calcium (a) and phosphorous (b) concentrations in the serum phase of 5% w/v SMP dispersions (5% w/v) before and after acidification to pH 2.4–3.0, before and after heating at 90 � C for 2 min. Different letters above bars indicate significant difference in the compared parameter (P < 0.05). Error bars are SD (n ¼ 3).
Fig. 9. Reducing (a,b) and non-reducing (c,d) SDS-PAGE of 5% w/v SMP dis persions before (pH 6.8, far right lane in each panel of four lanes) and after acidification to pH 2.4, 2.7, and 3.0 (left to right in each panel of four lanes) before (a,c) and after (b,d) heating at 90 � C for 2 min. 9
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(PAGE). As shown in Fig. 9(a–d), the intensity of each band was similar irrespective of pH and heating in both reducing and non-reducing PAGE. This is similar to another reducing SDS-PAGE study (Ranadheera et al., 2019) showing no apparent change in band intensity of milk protein concentrate dispersions between pH 2 and pH 4.6. Similar band in tensity and pattern of SMP dispersions suggest either little inter-molecular disulfide bonds formed after acidification and heating or the changes were not detectable at the studied conditions. Acidification from pH 6.8 to 2.4 before heating decreased the amount of free thiol groups (Fig. 10). At pH 6.8, β-Lg exists as dimers and is arranged as open conformation; however, lowering pH causes closed conformation of β-Lg by moving E-F loop (residues 86–89), resulting in less access to amino acid residues, i.e., free thiol groups (Uhrínov� a et al., 2000). The concentration of free thiol groups decreased significantly (P < 0.05) after heating samples at pH 6.8. Disulfide bonding due to sulfhydryl-disulfide exchange is the major mechanism governing the formation of whey protein-casein micelle complexes at pH 6.7 (Jang et al., 1990). This suggests that the decreased number of free thiol groups involves with disulfide bonding between whey proteins, partic ularly β-Lg, or between whey proteins and caseins, e.g., β-Lg and κ-casein after the heating at pH 6.8. At pH 3, the concentration of free thiol group did not change significantly after heating, which is in agreement with a previous study (Monahan, German, & Kinsella, 1995). No reduction in free thiol group content at acidic pH is expected, as formation of disulfide bonds is inhibited due to limited reactivity of free thiol groups at acidic pH (Alting, de Jongh, Visschers, & Simons, 2002). However, heating resulted in the increased free thiol groups at pH 2.4 and 2.7, possibly due to further dissociation of casein micelles causing the increased exposure of free thiol groups. Combining all the results above, however, there is still no clear evidence resulting a vivid band emerged at ~80 kDa on the reducing SDS-PAGE (Fig. 9a and b).
Fig. 10. Contents of free thiol groups in the 5% w/v SMP dispersions before and after acidification to pH 2.4–3.0, before and after subsequent heating at 90 � C for 2 min. Different letters above bars indicate significant difference (P < 0.05). Error bars are SD (n ¼ 3).
Declaration of competing interest We do not have any undisclosed relationship and funding source that may pose a competing interest. Acknowledgement The authors are grateful to Dr. John Dunlap, Dr. Michael Essington, Dr. Edward Wright, and Melanie Stewart for assisting with experiments. This study was supported by USDA National Institute of Food and Agriculture, TEN2015-05921 and hatch projects TEN00487 and 223984. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture.
4. Conclusion Acidification of SMP dispersion to pH 3.0 with citric acid increased the solubility of CCP and, together with calcium-chelating properties of citrate resulted in dissociation of casein micelles and reduction of dispersion turbidity. The dissociated caseins formed open fractal struc tures at pH 3.0, involving hydrophobic interactions, hydrogen bonds and cation-π interactions according to fluorescence spectroscopy, to result in the higher Dh than the SMP dispersion at pH 6.8, but the low refractive index of fractal structures was responsible for the lowered dispersion turbidity at pH 3.0. Upon further acidification to pH 2.7 and 2.4, the continued dissolution of CCP further reduced the dimension of casein micelles and, together with the reduced refractive index, resulted in the lower turbidity and smaller Dh than the SMP dispersion at pH 6.8. Upon heating at pH 2.4, no disulfide bond formation, based on SDSPAGE and free thiol content, and no significant changes in serum cal cium and phosphate concentrations were detected at the studied con ditions, and hydrophobic interactions between caseins were weakened to reduce the dimension of fractal structures to further reduce SMP dispersion turbidity and Dh. With extended heating at pH 2.4, whey protein denaturation was insignificant at 70 � C and the SMP dispersion showed insignificant changes in turbidity and Dh. However, whey pro tein denaturation was possible at 90 � C, and the calcium ions in the serum and the increased ionic strength relative to MC and MC/WPI dispersions may have facilitated the aggregation of proteins in SMP dispersions to cause the continued increase in Dh at a longer heating time. The open fractal structures with a low refractive index were also responsible for insignificant changes in SMP dispersion turbidity after extended heating at 90 � C. The exact mechanism of serum ions impacting structures of SMP dispersions however is to be further stud ied. Nevertheless, findings from the present study may be used to guide the incorporation of SMP in relevant translucent protein-fortified beverages.
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