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Effects of superfine grinding and microparticulation on the surface hydrophobicity of whey protein concentrate and its relation to emulsions stability
Food Hydrocolloids 51 (2015) 512e518
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Effects of superfine grinding and microparticulation on the surface hydrophobicity of whey protein concentrate and its relation to emulsions stability Chanchan Sun 1, Tao Wu 1, Rui Liu, Bin Liang, Zhaojie Tian, Enqi Zhang, Min Zhang* Key Laboratory of Food Nutrition and Safety (Tianjin University of Science & Technology), Ministry of Education, Tianjin 300457, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 January 2015 Received in revised form 18 May 2015 Accepted 22 May 2015 Available online 9 June 2015
In this study, superfine grinding and microparticulation were employed to increase the hydrophobicity of whey protein concentrate (WPC), which was investigated using 8-anilino-1-naphthalene sulfonic acid. WPC-stabilized emulsions containing 40% and 80% (w/w) oil showed smaller average droplet size when superfine grinding-treated WPC (sWPC) and microparticulated protein (MPP) were used than when WPC was used. The micro-rheology of the emulsions was measured upon diffusing wave spectroscopy; results showed that MPP can form stable emulsions. Moreover, the solid liquid balance of the emulsions significantly decreased from 0.87 to 0.38, with changing states from liquid to solid. The Turbiscan Stability Indexes of sWPC and MPP significantly reduced from 15.23 to 13.58 and 1.17, respectively. The operations of superfine grinding and microparticulation rendered WPC into an ingredient with excellent emulsifying properties. Thus, this ingredient can be utilized in both reduced-fat and high-fat food applications. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Whey protein concentrate Superfine grinding Microparticulation Surface hydrophobicity Microrheology Stability
1. Introduction Whey proteins (WPs) are widely used in the food industry because of their excellent functional and nutritional properties. WPs stabilize emulsions through their capability to adsorb at the oilewater interface. The final stability of emulsions before consumption is an important consideration for their utilization in the food industry. The formation and stability of emulsions largely depend on the molecular interactions of the adsorbed protein and how the interactions are influenced by environmental conditions, such as pH, ionic strength, or temperature (Mustapha, Ruttarattanamongkol, & Rizvi, 2012). Protein surface hydrophobicity is another important factor associated with emulsion stability, and researchers have realized its importance while explaining the surface functionality of proteins (Mitidieri & Wagner, 2002). Microparticulated whey protein (MWP) has been used as a fat replacer in the food industry to improve the sensory properties and
* Corresponding author. Tel.: þ86 022 60912341; fax: þ86 022 60912340. E-mail address: [email protected] (M. Zhang). 1 These authors contributed to the work equally and should be regarded as cofirst authors. http://dx.doi.org/10.1016/j.foodhyd.2015.05.027 0268-005X/© 2015 Elsevier Ltd. All rights reserved.
nutritional value of dairy products (Lo & Bastian, 1998), such as ice cream (Yilsay, Yilmaz, & Bayizit, 2006), yogurt (Torres, Janhøj, Mikkelsen, & Ipsen, 2011), and cheese (Ismail, Ammar, & ElMetwally, 2011). To date, the production of microparticulated protein (MPP) involves the application of heat and shear to the proteins (Lucca & Tepper, 1994). The combination of acidification and heat treatment in microparticulation improves the foaming and emulsifying properties of proteins because of an increase in surface hydrophobicity induced by deamidation and denaturation (Moro, Gatti, & Delorenzi, 2001). Superfine grinding has shown a great potential in producing nutraceuticals and functional foods (Chen, Weiss, & Shahidi, 2006). Some studies reported that superfine protein powder possesses high fluidity, solubility, electric conductivity, and water holding capacity; this finding indicates that superfine grinding improves the quality of food products (Wu, Zhang, Wang, Mothibe, & Chen, 2012; Zhao, Yang, Gai, & Yang, 2009). Nevertheless, insufficient information is available about the properties of superfine grindingtreated WPC (sWPC). Therefore, this study aims to investigate the effect of superfine grinding and microparticulation on surface hydrophobicity of WPC and its relation to the storage stability of sWPC and MWP-stabilized concentrated cold-set emulsion gels.
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2. Materials and methods 2.1. Materials Commercial WPC was purchased from Fonterra Commercial Trading (Shanghai) Co., Ltd. The WPC comprised 80.3% protein (wet basis), 3.5% lactose monohydrate, 3.4% starch, 3.8% fat, 5.1% moisture, and 3.9% ash. Rhodamine B and 8-anilino-1-naphthalene sulfonic acid (ANS) were obtained from SigmaeAldrich (St. Louis, MO, USA). Commercial soybean oil was purchased from a local store in Tianjin. All chemicals were reagent grade. 2.2. Production of sWPC through superfine grinding sWPC was ground in a multidimensional swing high-energy nanoball mill (CJM-SY-B Qinhuangdao Taiji Ring Nano-Products Co., Ltd., Hebei, China). Milling ball is composed of zirconia, and the ratio of milling ball and material is 6. To prevent heat denaturation, the working temperature was controlled at 35 C. In this study, sWPC-4h and sWPC-8h represent WPC treated through superfine grinding for 4 and 8 h, respectively.
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The protein solutions (0.05%, w/w protein) were prepared in 5 mM phosphate buffer (pH 7.0). The fluorescence emission spectra of tryptophan were obtained using an FL-2500 fluorescence spectrophotometer (Hitachi, Science Systems, Ibaraki, Japan). Fluorescence intensity was recorded at excitation and emission wavelengths of 295 and 300e400 nm, respectively. The excitation and emission slit widths were 5 and 5 nm, respectively, and the voltage was 700 mV. 2.7. Emulsion preparation Continuous-phase emulsions were prepared from a fixed concentration (12%, w/w) of proteins at pH 4.5 by stirring the appropriate amount of protein in deionized water for 2 h at room temperature (25 C) and then storing the mixture overnight at 4 C. Emulsions containing 40% and 80% (w/w) soybean oil were prepared by mixing appropriate amounts of protein solutions and oil in glass containers by using a high-speed dispersing unit (IKA Ultra Turrax, T25 basic, IKA Works, Inc., NC, USA) at 12,000 r/min for 3 min at room temperature (25 C). Sodium azide (0.02%, w/w) was added to prevent microbial growth. 2.8. Diffusing wave spectroscopy (DWS)
2.3. Production of MPP Approximately 0.12 g/mL protein solutions (pH 4.5) were heated at 85 C for 35 min and then homogenized in an ULTRA-TURRAX (IKA) T18 high-speed homogenizer for 6 min at 10,000 r/min. The obtained proteins were lyophilized to yield MPP. In this study, MWP represents microparticulated WPC, MPP-4h represents microparticulated sWPC-4h, and MPP-8h represents microparticulated sWPC-8h. 2.4. Determination of particle size distributions The particle size distributions of WPC, sWPC-4h, and sWPC-8h were determined using a BT-2001 Laser Analyzer (Dandong Bettersize Instruments Ltd., China), with air as a dispersion medium. The particle size distributions of MPP dispersions were determined using a BT-9300S Laser Analyzer (Dandong Bettersize Instruments Ltd., China), with water as a dispersion medium. The laser analyzers were all based on dynamic light scattering. The StokeseEinstein equation was used to calculate the particle size and distribution. 2.5. Protein surface hydrophobicity (PSH) index The PSH was determined through fluorescence spectroscopy with an ANS probe in accordance with the method described by Alizadeh-Pasdar and Li-Chan (2000) with slight modifications. The relative fluorescence intensity was obtained using an FL-2500 fluorescence spectrophotometer (Hitachi, Science Systems, Ibaraki, Japan). Fluorescence intensity was recorded at excitation and emission wavelengths of 340 and 500 nm, respectively. The excitation and emission slit widths were 2.5 and 2.5 nm, respectively. The index of surface hydrophobicity was determined from the initial slope of the plot of fluorescence intensity versus protein concentration. Within the protein concentration range used in these experiments, linear relationships were obtained (R2 ¼ 0.98e0.99).
The microrheology of emulsions was measured using Rheolaser Master (Formulaction, l'Union, France) through DWS. When a laser beam illuminates a fluid sample, the photons penetrating into the sample are backscattered by micro-objects, such as particles, droplets, and fibers, which are suspended in the fluid. A video camera was used to record the dynamic interference patterns of the backscattered waves, often known as “the speckle image.” Standard numerical algorithms are used to deduce the statistical parameters of the sample from dynamic speckle images; such parameters include MSD as a function of time (Chen et al., 2012). The MSD of these tracer particles is a direct and noninvasive probe of medium properties. Immediately after preparation, the emulsions were placed in flat-bottomed cylindrical glass tubes (140 mm, height; 16 mm, diameter), and the first measure of backscattered light intensity was performed. 2.9. Emulsion stability Emulsion stability was monitored through visual inspection with the optical scanning instrument Turbiscan ASG (Formulaction, l'Union, France) for kinetic stability studies. This instrument is composed of a detection head that moves up and down along a flatbottomed cylindrical cell. The detection head facilitates vertical scans of the entire length of the sample. For opaque systems such as emulsions, the apparatus is used to measure the backscattered light intensity of the sample as a function of sample height and time. The prepared emulsions were placed in flat-bottomed cylindrical glass tubes (140 mm, height; 16 mm, diameter), and the first measure of backscattered light intensity was performed. The tubes were stored at 25 C, and backscattered light was scanned every hour for 24 h. In this part, the dynamic of stability curve di was calculated using Eq. (1) to draw a di portrait along the time:
PH
h¼0 jscann ðhÞ
scann1 ðhÞj
2.6. Steady-state fluorescence spectra
dn ¼
Most proteins contain intrinsically fluorescent amino acid residues, such as tryptophan, tyrosine, and phenylalanine. Tryptophan is by far the most useful of these amino acid residues. Royer (2006) reported that specific local information can be attained by selectively exciting the tryptophan residues at 295 nm or above.
where n is the number of scanning, scan is the light intensity, h is the height per 40 mm, and H is the height of the samples in the cell. The Turbiscan Stability Index (TSI) is a statistical parameter used to estimate the suspension stability (Wisniewska, 2010). The TSI was obtained as the sum of all processes occurring in the studied
H
(1)
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probe. The TSI values were calculated using Eq. (2) with a specific computer program:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Pn i¼1 xi xBS TSI ¼ n1
(2)
where xi is the backscattering for each minute of measurement, xBS is the mean xi, and n is the number of scans. 2.10. Droplet size distributions of the emulsions The size distribution of the emulsion droplets was measured using a BT-9300S laser analyzer (Dandong Bettersize Instruments Ltd., China). The diffraction of laser light by a diluted portion of the emulsion was detected on an array of detectors and transformed into size distribution by the software. 2.11. Statistical analysis Tests were performed in triplicate. The illustrated figures are one of the parallel experiments, and the results are presented as mean ± SD. Results were evaluated using one-way ANOVA, followed by Student's test for statistical analysis. Differences were considered statistically significant at p < 0.05. 3. Results and discussion 3.1. Particle size distribution Fig. 1 shows that all the particle sizes presented normal distribution (unimodal distribution). Hence, the mean particle size (D50) was selected to characterize protein size (Table 1). The mean particle sizes of sWPC-4h and sWPC-8h decreased from 51.1 mm (WPC) to 14.5 and 8.0 mm after superfine grinding, respectively. The mean particle sizes of MPP accordingly decreased from 13.3 mm to 10.8 and 8.9 mm. The decrease in particle size favors the production and application of microparticulated protein. 3.2. PSH Table 2 illustrates the changes in aromatic hydrophobicity after superfine grinding and microparticulation. The results of ANS showed that the PSH of sWPC was significantly (p < 0.05) higher
Table 1 Median particle size (D50) of samples. The values that do not bear the same letter in the same column are significantly different (p < 0.05). Powers
D50 (mm)
MPP
D50 (mm)
WPC sWPC-4h sWPC-8h
51.1 ± 5.5a 14.5 ± 1.3b 8.0 ± 0.3bc
MWP MPP-4h MPP-8h
13.3 ± 0.1a 10.8 ± 0.1b 8.9 ± 0.1c
than that of WPC and increased as the time of superfine grinding treatment was prolonged. The PSH values of MPP-4h and MPP-8h were significantly (p < 0.05) higher than that of MWP and increased as the time of superfine grinding treatment was prolonged. The findings reflected a significant change in ANS access to the hydrophobic sites on the protein molecules as a result of the modification process. This phenomenon may be attributed to the changes in the conformation of b-lactoglobulin (b-Lg) that contains a high proportion of hydrophobic amino acid side chains (Laligant, Dumay, Casas Valencia, Cuq, & Cheftel, 1991). Meanwhile, the PSH values of microparticulated proteins were significantly (p < 0.05) higher than those of WPC and sWPC (Table 2). Alizadeh-Pasdar and Li-Chan (2000) observed that the thermal denaturation of b-Lg, WPC, and WPI solutions heated (above 70 C) at acidic and neutral pH increases the aromatic surface hydrophobicity of the proteins. Furthermore, hydrophilic groups combined with water molecules and were buried inside the heated aggregation. The diminishing surface hydrophilic groups also increased the chance of ANS binding with aromatic hydrophobicity. Therefore, the combined effect of intense shear during superfine grinding and heat during microparticulation transformed the protein conformations. This phenomenon exposed the aromatic hydrophobic amino acids. 3.3. Steady-state fluorescence spectra Fig. 2A suggests that the maximum emission wavelength of WPC occurred at 333 nm. The maximum emission wavelengths of sWPC-4h and sWPC-8h shifted to longer wavelengths at 334 nm. After microparticulation, the maximum emission wavelength shifted to longer wavelength at 356 nm, and MPP-4h and MPP-8h continued to redshift to 358 nm (Fig. 2B). Given its aromatic character, tryptophan is often (although not always) found completely or partially buried in the hydrophobic core of protein interiors, at the interface between two
Fig. 1. Particle size distributions of proteins (A: powers; B: MPP).
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Table 2 Protein surface hydrophobicity (PSH) of samples. The values that do not bear the same letter in the same row are significantly different (p < 0.05). Samples
WPC
MWP
sWPC-4h
MPP-4h
sWPC-8h
MPP-8h
PSH
99.8 ± 10.2f
231.5 ± 16.8c
151.0 ± 3.5e
320.2 ± 2.5b
175.7 ± 7.2d
561.2 ± 13.3a
Fig. 2. Fluorescence emission spectrum of tryptophan.
protein domains or subdomains, or at the subunit interface in oligomeric protein systems. Disruption of the tertiary or quaternary structure of the protein further exposed the side chains to the solvent. In general, the exposure of the hydrophobic group can affect the ground- and excited-state energy levels of the chromophores, reduce the energy of the excited state, and cause the redshift of the emission spectrum (increased lambda Max) (Chan, Pinotsi, Kaminski Schierle, & Kaminski, 2014). In the present study, the redshift of the maximum emission wavelength of superfine and microparticulated proteins indicates the exposure of the hydrophobic group. This finding is consistent with the increased PSH of the proteins after superfine grinding and microparticulation. 3.4. Microrheology Figs. S1 and S2 illustrate the MSD curves at different aging times for the emulsions containing 40% and 80% oil. The measurements were started after sample preparation. The curves were displaced to larger time scales and developed more curvature with increasing oleaginousness, indicating increased viscosity and storage modulus, respectively (Pinder, Swanson, Hebraud, & Hemar, 2006). As illustrated in Fig. S1A1, B1, and C1, no significant movement was observed. This result indicates that the emulsions stabilized. The MSD curves moved from long to short as the superfine grinding time was prolonged. This result indicates increased elasticity. The same trend was observed for MWP (Fig. S2A1), MPP4h (Fig. S2B1), and MPP-8h (Fig. S2C1) emulsions containing 40% oil. However, when the oil content was increased to 80%, the sample showed the typical characteristic of a purely viscous product. That is, the MSD linearly increased with decorrelation time as the particles became completely free to move in the samples [Fig. S1A2, B2, and C2]. The MSD curves initially moved from long to short displacement (indicating increased elasticity) and from short to long decorrelation time (indicating increased braud, viscosity), and then stabilized (Bellour, Skouri, Munch, & He 2002). This step monitors network flocculation and stabilization.
Fig. S2A2, B2, and C2 shows the typical shape for the MSD of a viscoelastic product (concentrated emulsion, polymer solution with particles). Within short observation times, the particles can freely move in the continuous phase. The particles were blocked by their neighbors (or by polymers), and the MSD reached a plateau. This result is characteristic of product elasticity. Lower plateau resulted in tighter network and stronger elasticity. As the time scales were prolonged, the scatterers escaped from the “cages” formed by neighboring particles or polymers, and the MSD grew as it would for a viscous fluid. This result is characteristic of macroscopic viscosity because it corresponds to the speed of particles in the sample. In summary, the MSD is the viscoelastic fingerprint of the analyzed product. Table 3 shows the solideliquid balance (SLB), which corresponds to the MSD slope at short decorrelation time and represents the domination status of the sample liquid or solid. SLB ¼ 0.5 is a critical value of the transformation from liquid domination to solid domination. According to Tisserand, Fleury, Brunel, Bru, and Meunier (2012), the SLBs of WPC, sWPC-4h, and sWPC-8h emulsions containing 40% oil are greater than 0.5 and less than 1. This finding indicates that the liquid dominates the dispersions. Nevertheless, the SLBs of other emulsions were all smaller than 0.5. This finding indicates that the solid dominates the dispersions (gel behavior). Overall, the SLBs of sWPC-4h, sWPC-8h, MWP, MPP-4h, and MPP-8h emulsions containing the same amount of oil were significantly lower than that of WPC emulsion. Lower elastic plateau resulted in stronger elasticity. The elasticity index (EI) corresponds to the inverse of the MSD plateau value. As shown in Table 3, the EIs of sWPC-4h, sWPC-8h, MWP, MPP-4h, and MPP-8h emulsions containing the same amount of oil were significantly higher than that of WPC emulsion. This finding indicates increased elasticity. The macroscopic viscosity index (MVI), which corresponds to the inversed MSD slope in linear scale, is linked to the well-known macroscopic viscosity in Pa s. As shown in Table 3, the MVIs of sWPC-4h, sWPC-8h, MWP, MPP-4h, and MPP-8h emulsions containing the same amount of oil were significantly lower than that of WPC emulsion. This result indicates decreased viscosity. According
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Table 3 Solid liquid balance (SLB), elasticity index (EI) and macroscopic viscosity index (MVI) of emulsions at 10 min. The values that do not bear the same letter in the same column are significantly different (p < 0.05). Samples 40%
80%
WPC MWP sWPC-4h MPP-4h sWPC-8h MPP-8h WPC MWP sWPC-4h MPP-4h sWPC-8h MPP-8h
EI (103, nm2)
SLB 0.87 0.44 0.81 0.43 0.68 0.40 0.49 0.44 0.47 0.42 0.40 0.38
± ± ± ± ± ± ± ± ± ± ± ±
a
0.03 0.01fg 0.02b 0.01fh 0.02c 0.01ijk 0.02d 0.01f 0.02 de 0.01fghijk 0.01i 0.01ijl
1.15 1.10 1.14 1.63 1.18 1.71 2.86 17.50 2.85 67.23 1.68 160.37
± ± ± ± ± ± ± ± ± ± ± ±
ij
0.05 0.03ijkl 0.05ijk 0.05fgh 0.06i 0.02f 0.06d 0.17c 0.11de 1.56b 0.01fg 9.69a
MVI (104, nm2 s) 0.17 144.33 0.18 18.74 0.41 6.49 123.00 671.77 199.07 444.30 9.92 280.27
± ± ± ± ± ± ± ± ± ± ± ±
0.06ijkl 10.53e 0.02ijk 1.65g 0.08ij 0.02hi 8.61f 14.02a 13.70d 24.03b 1.13h 13.52c
to Mason (2000), higher macroscopic viscosity means more time is needed by the particles to travel a distance. Therefore, they have a lower particle speed, which may result in enhanced emulsion stability. 3.5. Emulsion stability The TSI curves of emulsions containing 40% oil showed a significant increasing trend, except for MPP-8h emulsions (Fig. 3A). The TSI curves of MPP emulsions containing 80% oil and MPP-8h emulsions containing 40% oil were flat and low (Fig. 3B). This result indicates high stability (Durand, Franks, & Hosken, 2003). The high TSI values can be attributed to the precipitation of protein and oil on the bottom and top of the vial, respectively (Wisniewska et al., 2013). Stable emulsions were defined as those that showed no significant changes in the oil droplet size and/or had no creaming or oiling off after storage. Table 4 shows the sum TSI of the emulsions at 24 h; this parameter reflects the storage stability of the emulsions. The TSI of the emulsions decreased as the time of superfine grinding was prolonged and after microparticulation. A decrease in TSI corresponded to an increase in stability. Thus, the stability of protein emulsions containing 40% and 80% oil increased after superfine grinding and microparticulation treatment. Similar findings were demonstrated by Dissanayake and Vasiljevic (2009) who postulated that the partial unraveling of WPs allows a thermodynamically favorable conformation for adherence to the watereoil interface.
A decrease in TSI corresponded to increases in PSH, MVI, EI, and oil level. On the basis of these findings, the enhanced aromatic hydrophobicity, MVI, EI, and oil levels were proposed to be the main factors regulating the stability of the MPP-8h emulsions upon storage. The combined effects of the first two factors predominantly affected the stability of the emulsions against creaming. The larger number of exposed aromatic hydrophobic residues increased the affinity of the protein molecules toward the oilewater interface. This phenomenon enhanced the proteinelipid interaction and simultaneously shortened the time required to reduce the surface tension, thereby producing small droplets. Furthermore, the large surface hydrophobicity may enhance the interaction between adjacent protein molecules that adsorbed at the interface, thereby forming a rigid and thick protein membrane that provides a considerable steric stabilization to the emulsion droplets. The enhanced emulsification of heat-denatured soy proteins and WPC possibly results from the exposure of buried hydrophobic groups (Surh, Gu, Decker, & McClements, 2005). The enhanced MVI of the continuous phase allowed the formation of a weak gel matrix that entrapped the fat droplets within the network, whereas increased oil levels enhanced the interactions between the droplets and protein; as a result, the movement of the droplets was retarded (Rosa, Sala, Van Vliet, & Van De Velde, 2006). 3.6. Droplet size distributions of the emulsions Fig. 4 shows the droplet size distributions of the emulsions containing 40% and 80% oil at 0 h. All of the droplet sizes of WPC and MWP emulsions containing 40% oil presented normal distribution (unimodal distribution) within approximately 15 mm (Fig. 4A). The droplet size distribution of sWPC-4h, sWPC-8h, MPP4h, and MPP-8h emulsions containing 40% presented another peak at approximately 3 mm. In addition, the peak height at approximately 3 mm increased as the time of superfine grinding treatment was prolonged. This result indicates that the number of small droplets increased. Meanwhile, the vertex ordinate indistinctively shifted to a smaller droplet size, which means the diminution of the droplet size with superfine grinding treatment. The same tendency was observed for the droplet size distributions of emulsions containing 80% oil. The conclusions are in accordance with the tendency of the mean droplet size (D50) (Table 5). Therefore, D50 was selected to intuitively characterize the droplet sizes.
Fig. 3. Turbiscan stability index (TSI) of emulsions containing (A) 40% and (B) 80% oil.
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Table 4 The Turbiscan stability index (TSI) of emulsions at 24 h ðx±SDÞ. The values that do not bear the same letter in the same row are significantly different (p < 0.05). Oil levels (%)
TSI WPC
40% 80%
MWP a
15.23 ± 0.35 11.19 ± 0.67a
sWPC-4h ab
14.55 ± 0.51 2.87 ± 0.44e
MPP-4h abc
14.88 ± 0.41 9.75 ± 0.94ab
sWPC-8h abce
12.06 ± 1.23 6.35 ± 0.29cd
MPP-8h cd
13.58 ± 0.68 8.53 ± 0.75bc
6.24 ± 1.26f 1.17 ± 0.02ef
Fig. 4. Droplet size distributions of the emulsions (A: emulsions containing 40% oil; B: emulsions containing 80% oil).
Table 5 Median particle size (D50) of the emulsion droplets. The values that do not bear the same letter in the same row are significantly different (p < 0.05). Oil levels (%)
40% 80%
D50 (mm) WPC
MWP
sWPC-4h
MPP-4h
sWPC-8h
MPP-8h
16.2 ± 0.1a 13.5 ± 1.0a
11.1 ± 1.5bcde 9.3 ± 1.2bc
13.4 ± 0.6ab 9.5 ± 0.5ef
11.3 ± 0.3bd 10.0 ± 0.2b
12.1 ± 3.0bcd 9.2 ± 0.5bcd
7.9 ± 1.0def 8.5 ± 0.3bcde
No significant difference in D50 was observed between WPC and sWPC-4h emulsions containing 40% and 80% oil. However, the D50 of sWPC-8h emulsions containing 40% and 80% oil was significantly lower (p < 0.05) than that of the emulsions containing the same levels of oil. The observed behavior can be ascribed to the increased adsorbance efficiency of sWPC-8h onto the oilewater interface. In general, the fat droplet size decreased with increasing oil levels regardless of protein type. This result may be attributed to the fact that the emulsions were constructed by a loose network of spherical droplets that progressed to form a compact matrix as the oil level was increased to 80%. The findings agreed with the observation by Hemar and Horne (2000) for caseinate-stabilized
emulsions in which the spherical, close packing limit was shown to fall around 70% oil. The authors reported hexagonal and pentagonal structures in emulsions with a highly dispersed phase volume. Therefore, the high emulsibility of the sWPC- and MPPstabilized emulsions can be advantageously exploited in the manufacture of spread-like reduced-fat and gel-like high-fat foods. 4. Conclusions Superfine grinding and microparticulation were employed to produce new WPC ingredients, namely, sWPC and MPP. The redshift of the maximum emission wavelength (from 333 nm to
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334 nm, 356 nm, and 358 nm) and the increase in PSH indicated an increase the hydrophobicity of WPC and changes in the microenvironment of the fluorophore inside the protein molecule. The decrease in SLB and the increases in EI and MVI were attributed to the increased ratio of oil phase, superfine grinding treatment, and microparticulation. This result indicates that the status of the emulsions changed from liquid to viscoelastic behavior. The TSI of the emulsions at 24 h directly reflected the higher emulsibility of sWPC, and the use of MPP as the continuous phase resulted in concentrated emulsions containing 40% and 80% oil. These emulsions remained stable even after prolonged storage of 24 h at 25 C. These findings imply that enhanced PSH, MVI, EI, and higher oil level are the main factors regulating the stability of stabilized emulsions upon storage. The results of this study confirmed our hypothesis that reactive superfine grinding and microparticulation render WPC into an ingredient with excellent gelling and emulsifying properties. This finding may widen the applications of sWPC and MPP in food emulsions. sWPC and MPP with an excellent emulsion-stabilizing property can be utilized in both reduced-fat and high-fat food applications requiring high stability. Acknowledgments The authors acknowledge the financial support from the National High Technology Research and Development Program of China (863 Program) (Grant no. 2013AA102204). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.foodhyd.2015.05.027. References Alizadeh-Pasdar, N., & Li-Chan, E. C. (2000). Comparison of protein surface hydrophobicity measured at various pH values using three different fluorescent probes. Journal of Agricultural and Food Chemistry, 48(2), 328e334. braud, P. (2002). Brownian motion of Bellour, M., Skouri, M., Munch, J.-P., & He particles embedded in a solution of giant micelles. The European Physical Journal E: Soft Matter and Biological Physics, 8(4), 431e436. Chan, F. T., Pinotsi, D., Kaminski Schierle, G., & Kaminski, C. F. (2014). Structurespecific intrinsic fluorescence of protein amyloids used to study their kinetics of aggregation. Academic Press. Chen, H., Weiss, J., & Shahidi, F. (2006). Nanotechnology in nutraceuticals and functional foods. Food Technology, 60(3), 30e36. Chen, Y. Q., Chou, P.l., Cheng, C. Y., Chiang, C. C., Wei, M. T., Chuang, C. T., et al. (2012). Microrheology of human synovial fluid of arthritis patients studied by diffusing wave spectroscopy. Journal of Biophotonics, 5(10), 777e784. Dissanayake, M., & Vasiljevic, T. (2009). Functional properties of whey proteins affected by heat treatment and hydrodynamic high-pressure shearing. Journal of Dairy Science, 92(4), 1387e1397.
Durand, A., Franks, G., & Hosken, R. (2003). Particle sizes and stability of UHT bovine, cereal and grain milks. Food Hydrocolloids, 17(5), 671e678. Hemar, Y., & Horne, D. (2000). Dynamic rheological properties of highly concentrated protein-stabilized emulsions. Langmuir, 16(7), 3050e3057. Ismail, M., Ammar, E. T., & El-Metwally, R. (2011). Improvement of low fat mozzarella cheese properties using denatured whey protein. International Journal of Dairy Technology, 64(2), 207e217. Laligant, A., Dumay, E., Casas Valencia, C., Cuq, J. L., & Cheftel, J. C. (1991). Surface hydrophobicity and aggregation of beta-lactoglobulin heated near neutral pH. Journal of Agricultural and Food Chemistry, 39(12), 2147e2155. Lo, C., & Bastian, E. (1998). Incorporation of native and denatured whey proteins into cheese curd for manufacture of reduced fat, Havarti-type cheese. Journal of Dairy Science, 81(1), 16e24. Lucca, P. A., & Tepper, B. J. (1994). Fat replacers and the functionality of fat in foods. Trends in Food Science & Technology, 5(1), 12e19. Mason, T. G. (2000). Estimating the viscoelastic moduli of complex fluids using the generalized StokeseEinstein equation. Rheologica Acta, 39(4), 371e378. Mitidieri, F. E., & Wagner, J. R. (2002). Coalescence of o/w emulsions stabilized by whey and isolate soybean proteins. Influence of thermal denaturation, salt addition and competitive interfacial adsorption. Food Research International, 35(6), 547e557. Moro, A., Gatti, C., & Delorenzi, N. (2001). Hydrophobicity of whey protein concentrates measured by fluorescence quenching and its relation with surface functional properties. Journal of agricultural and food chemistry, 49, 4784e4789. Mustapha, N. A., Ruttarattanamongkol, K., & Rizvi, S. S. H. (2012). The effects of supercritical fluid extrusion process on surface hydrophobicity of whey protein concentrate and its relation to storage and heat stability of concentrated emulsions. Food Research International, 48(2), 470e477. Pinder, D., Swanson, A., Hebraud, P., & Hemar, Y. (2006). Micro-rheological investigation of dextran solutions using diffusing wave spectroscopy. Food Hydrocolloids, 20(2), 240e244. Rosa, P., Sala, G., Van Vliet, T., & Van De Velde, F. (2006). Cold gelation of whey protein emulsions. Journal of Texture Studies, 37(5), 516e537. Royer, C. A. (2006). Probing protein folding and conformational transitions with fluorescence. Chemical reviews, 106(5), 1769e1784. Surh, J., Gu, Y. S., Decker, E. A., & McClements, D. J. (2005). Influence of environmental stresses on stability of O/W emulsions containing cationic droplets stabilized by SDSefish gelatin membranes. Journal of Agricultural and Food Chemistry, 53(10), 4236e4244. Tisserand, C., Fleury, M., Brunel, L., Bru, P., & Meunier, G. (2012). Passive microrheology for measurement of the concentrated dispersions stability. UK colloids 2011 (pp. 101e105). Springer. Torres, I. C., Janhøj, T., Mikkelsen, B.Ø., & Ipsen, R. (2011). Effect of microparticulated whey protein with varying content of denatured protein on the rheological and sensory characteristics of low-fat yoghurt. International Dairy Journal, 21(9), 645e655. Wisniewska, M. (2010). Influences of polyacrylic acid adsorption and temperature on the alumina suspension stability. Powder Technology, 198(2), 258e266. Wisniewska, M., Terpiłowski, K., Chibowski, S., Urban, T., Zarko, V. I., & Gun'ko, V. M. (2013). Effect of solution pH on the stability of mixed silica-alumina suspension in the presence of polyacrylic acid (PAA) with different molecular weights. Central European Journal of Chemistry, 11(1), 101e110. Wu, G. C., Zhang, M., Wang, Y. Q., Mothibe, K. J., & Chen, W. X. (2012). Production of silver carp bone powder using superfine grinding technology: suitable production parameters and its properties. Journal of Food Engineering, 109(4), 730e735. € Yilmaz, L., & Bayizit, A. A. (2006). The effect of using a whey protein fat Yilsay, T.O., replacer on textural and sensory characteristics of low-fat vanilla ice cream. European Food Research and Technology, 222(1e2), 171e175. Zhao, X., Yang, Z., Gai, G., & Yang, Y. (2009). Effect of superfine grinding on properties of ginger powder. Journal of Food Engineering, 91(2), 217e222.
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Effects of superfine grinding and microparticulation on the surface hydrophobicity of whey protein concentrate and its relation to emulsions stability Chanchan Sun 1, Tao Wu 1, Rui Liu, Bin Liang, Zhaojie Tian, Enqi Zhang, Min Zhang* Key Laboratory of Food Nutrition and Safety (Tianjin University of Science & Technology), Ministry of Education, Tianjin 300457, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 January 2015 Received in revised form 18 May 2015 Accepted 22 May 2015 Available online 9 June 2015
In this study, superfine grinding and microparticulation were employed to increase the hydrophobicity of whey protein concentrate (WPC), which was investigated using 8-anilino-1-naphthalene sulfonic acid. WPC-stabilized emulsions containing 40% and 80% (w/w) oil showed smaller average droplet size when superfine grinding-treated WPC (sWPC) and microparticulated protein (MPP) were used than when WPC was used. The micro-rheology of the emulsions was measured upon diffusing wave spectroscopy; results showed that MPP can form stable emulsions. Moreover, the solid liquid balance of the emulsions significantly decreased from 0.87 to 0.38, with changing states from liquid to solid. The Turbiscan Stability Indexes of sWPC and MPP significantly reduced from 15.23 to 13.58 and 1.17, respectively. The operations of superfine grinding and microparticulation rendered WPC into an ingredient with excellent emulsifying properties. Thus, this ingredient can be utilized in both reduced-fat and high-fat food applications. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Whey protein concentrate Superfine grinding Microparticulation Surface hydrophobicity Microrheology Stability
1. Introduction Whey proteins (WPs) are widely used in the food industry because of their excellent functional and nutritional properties. WPs stabilize emulsions through their capability to adsorb at the oilewater interface. The final stability of emulsions before consumption is an important consideration for their utilization in the food industry. The formation and stability of emulsions largely depend on the molecular interactions of the adsorbed protein and how the interactions are influenced by environmental conditions, such as pH, ionic strength, or temperature (Mustapha, Ruttarattanamongkol, & Rizvi, 2012). Protein surface hydrophobicity is another important factor associated with emulsion stability, and researchers have realized its importance while explaining the surface functionality of proteins (Mitidieri & Wagner, 2002). Microparticulated whey protein (MWP) has been used as a fat replacer in the food industry to improve the sensory properties and
* Corresponding author. Tel.: þ86 022 60912341; fax: þ86 022 60912340. E-mail address: [email protected] (M. Zhang). 1 These authors contributed to the work equally and should be regarded as cofirst authors. http://dx.doi.org/10.1016/j.foodhyd.2015.05.027 0268-005X/© 2015 Elsevier Ltd. All rights reserved.
nutritional value of dairy products (Lo & Bastian, 1998), such as ice cream (Yilsay, Yilmaz, & Bayizit, 2006), yogurt (Torres, Janhøj, Mikkelsen, & Ipsen, 2011), and cheese (Ismail, Ammar, & ElMetwally, 2011). To date, the production of microparticulated protein (MPP) involves the application of heat and shear to the proteins (Lucca & Tepper, 1994). The combination of acidification and heat treatment in microparticulation improves the foaming and emulsifying properties of proteins because of an increase in surface hydrophobicity induced by deamidation and denaturation (Moro, Gatti, & Delorenzi, 2001). Superfine grinding has shown a great potential in producing nutraceuticals and functional foods (Chen, Weiss, & Shahidi, 2006). Some studies reported that superfine protein powder possesses high fluidity, solubility, electric conductivity, and water holding capacity; this finding indicates that superfine grinding improves the quality of food products (Wu, Zhang, Wang, Mothibe, & Chen, 2012; Zhao, Yang, Gai, & Yang, 2009). Nevertheless, insufficient information is available about the properties of superfine grindingtreated WPC (sWPC). Therefore, this study aims to investigate the effect of superfine grinding and microparticulation on surface hydrophobicity of WPC and its relation to the storage stability of sWPC and MWP-stabilized concentrated cold-set emulsion gels.
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2. Materials and methods 2.1. Materials Commercial WPC was purchased from Fonterra Commercial Trading (Shanghai) Co., Ltd. The WPC comprised 80.3% protein (wet basis), 3.5% lactose monohydrate, 3.4% starch, 3.8% fat, 5.1% moisture, and 3.9% ash. Rhodamine B and 8-anilino-1-naphthalene sulfonic acid (ANS) were obtained from SigmaeAldrich (St. Louis, MO, USA). Commercial soybean oil was purchased from a local store in Tianjin. All chemicals were reagent grade. 2.2. Production of sWPC through superfine grinding sWPC was ground in a multidimensional swing high-energy nanoball mill (CJM-SY-B Qinhuangdao Taiji Ring Nano-Products Co., Ltd., Hebei, China). Milling ball is composed of zirconia, and the ratio of milling ball and material is 6. To prevent heat denaturation, the working temperature was controlled at 35 C. In this study, sWPC-4h and sWPC-8h represent WPC treated through superfine grinding for 4 and 8 h, respectively.
513
The protein solutions (0.05%, w/w protein) were prepared in 5 mM phosphate buffer (pH 7.0). The fluorescence emission spectra of tryptophan were obtained using an FL-2500 fluorescence spectrophotometer (Hitachi, Science Systems, Ibaraki, Japan). Fluorescence intensity was recorded at excitation and emission wavelengths of 295 and 300e400 nm, respectively. The excitation and emission slit widths were 5 and 5 nm, respectively, and the voltage was 700 mV. 2.7. Emulsion preparation Continuous-phase emulsions were prepared from a fixed concentration (12%, w/w) of proteins at pH 4.5 by stirring the appropriate amount of protein in deionized water for 2 h at room temperature (25 C) and then storing the mixture overnight at 4 C. Emulsions containing 40% and 80% (w/w) soybean oil were prepared by mixing appropriate amounts of protein solutions and oil in glass containers by using a high-speed dispersing unit (IKA Ultra Turrax, T25 basic, IKA Works, Inc., NC, USA) at 12,000 r/min for 3 min at room temperature (25 C). Sodium azide (0.02%, w/w) was added to prevent microbial growth. 2.8. Diffusing wave spectroscopy (DWS)
2.3. Production of MPP Approximately 0.12 g/mL protein solutions (pH 4.5) were heated at 85 C for 35 min and then homogenized in an ULTRA-TURRAX (IKA) T18 high-speed homogenizer for 6 min at 10,000 r/min. The obtained proteins were lyophilized to yield MPP. In this study, MWP represents microparticulated WPC, MPP-4h represents microparticulated sWPC-4h, and MPP-8h represents microparticulated sWPC-8h. 2.4. Determination of particle size distributions The particle size distributions of WPC, sWPC-4h, and sWPC-8h were determined using a BT-2001 Laser Analyzer (Dandong Bettersize Instruments Ltd., China), with air as a dispersion medium. The particle size distributions of MPP dispersions were determined using a BT-9300S Laser Analyzer (Dandong Bettersize Instruments Ltd., China), with water as a dispersion medium. The laser analyzers were all based on dynamic light scattering. The StokeseEinstein equation was used to calculate the particle size and distribution. 2.5. Protein surface hydrophobicity (PSH) index The PSH was determined through fluorescence spectroscopy with an ANS probe in accordance with the method described by Alizadeh-Pasdar and Li-Chan (2000) with slight modifications. The relative fluorescence intensity was obtained using an FL-2500 fluorescence spectrophotometer (Hitachi, Science Systems, Ibaraki, Japan). Fluorescence intensity was recorded at excitation and emission wavelengths of 340 and 500 nm, respectively. The excitation and emission slit widths were 2.5 and 2.5 nm, respectively. The index of surface hydrophobicity was determined from the initial slope of the plot of fluorescence intensity versus protein concentration. Within the protein concentration range used in these experiments, linear relationships were obtained (R2 ¼ 0.98e0.99).
The microrheology of emulsions was measured using Rheolaser Master (Formulaction, l'Union, France) through DWS. When a laser beam illuminates a fluid sample, the photons penetrating into the sample are backscattered by micro-objects, such as particles, droplets, and fibers, which are suspended in the fluid. A video camera was used to record the dynamic interference patterns of the backscattered waves, often known as “the speckle image.” Standard numerical algorithms are used to deduce the statistical parameters of the sample from dynamic speckle images; such parameters include MSD as a function of time (Chen et al., 2012). The MSD of these tracer particles is a direct and noninvasive probe of medium properties. Immediately after preparation, the emulsions were placed in flat-bottomed cylindrical glass tubes (140 mm, height; 16 mm, diameter), and the first measure of backscattered light intensity was performed. 2.9. Emulsion stability Emulsion stability was monitored through visual inspection with the optical scanning instrument Turbiscan ASG (Formulaction, l'Union, France) for kinetic stability studies. This instrument is composed of a detection head that moves up and down along a flatbottomed cylindrical cell. The detection head facilitates vertical scans of the entire length of the sample. For opaque systems such as emulsions, the apparatus is used to measure the backscattered light intensity of the sample as a function of sample height and time. The prepared emulsions were placed in flat-bottomed cylindrical glass tubes (140 mm, height; 16 mm, diameter), and the first measure of backscattered light intensity was performed. The tubes were stored at 25 C, and backscattered light was scanned every hour for 24 h. In this part, the dynamic of stability curve di was calculated using Eq. (1) to draw a di portrait along the time:
PH
h¼0 jscann ðhÞ
scann1 ðhÞj
2.6. Steady-state fluorescence spectra
dn ¼
Most proteins contain intrinsically fluorescent amino acid residues, such as tryptophan, tyrosine, and phenylalanine. Tryptophan is by far the most useful of these amino acid residues. Royer (2006) reported that specific local information can be attained by selectively exciting the tryptophan residues at 295 nm or above.
where n is the number of scanning, scan is the light intensity, h is the height per 40 mm, and H is the height of the samples in the cell. The Turbiscan Stability Index (TSI) is a statistical parameter used to estimate the suspension stability (Wisniewska, 2010). The TSI was obtained as the sum of all processes occurring in the studied
H
(1)
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probe. The TSI values were calculated using Eq. (2) with a specific computer program:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Pn i¼1 xi xBS TSI ¼ n1
(2)
where xi is the backscattering for each minute of measurement, xBS is the mean xi, and n is the number of scans. 2.10. Droplet size distributions of the emulsions The size distribution of the emulsion droplets was measured using a BT-9300S laser analyzer (Dandong Bettersize Instruments Ltd., China). The diffraction of laser light by a diluted portion of the emulsion was detected on an array of detectors and transformed into size distribution by the software. 2.11. Statistical analysis Tests were performed in triplicate. The illustrated figures are one of the parallel experiments, and the results are presented as mean ± SD. Results were evaluated using one-way ANOVA, followed by Student's test for statistical analysis. Differences were considered statistically significant at p < 0.05. 3. Results and discussion 3.1. Particle size distribution Fig. 1 shows that all the particle sizes presented normal distribution (unimodal distribution). Hence, the mean particle size (D50) was selected to characterize protein size (Table 1). The mean particle sizes of sWPC-4h and sWPC-8h decreased from 51.1 mm (WPC) to 14.5 and 8.0 mm after superfine grinding, respectively. The mean particle sizes of MPP accordingly decreased from 13.3 mm to 10.8 and 8.9 mm. The decrease in particle size favors the production and application of microparticulated protein. 3.2. PSH Table 2 illustrates the changes in aromatic hydrophobicity after superfine grinding and microparticulation. The results of ANS showed that the PSH of sWPC was significantly (p < 0.05) higher
Table 1 Median particle size (D50) of samples. The values that do not bear the same letter in the same column are significantly different (p < 0.05). Powers
D50 (mm)
MPP
D50 (mm)
WPC sWPC-4h sWPC-8h
51.1 ± 5.5a 14.5 ± 1.3b 8.0 ± 0.3bc
MWP MPP-4h MPP-8h
13.3 ± 0.1a 10.8 ± 0.1b 8.9 ± 0.1c
than that of WPC and increased as the time of superfine grinding treatment was prolonged. The PSH values of MPP-4h and MPP-8h were significantly (p < 0.05) higher than that of MWP and increased as the time of superfine grinding treatment was prolonged. The findings reflected a significant change in ANS access to the hydrophobic sites on the protein molecules as a result of the modification process. This phenomenon may be attributed to the changes in the conformation of b-lactoglobulin (b-Lg) that contains a high proportion of hydrophobic amino acid side chains (Laligant, Dumay, Casas Valencia, Cuq, & Cheftel, 1991). Meanwhile, the PSH values of microparticulated proteins were significantly (p < 0.05) higher than those of WPC and sWPC (Table 2). Alizadeh-Pasdar and Li-Chan (2000) observed that the thermal denaturation of b-Lg, WPC, and WPI solutions heated (above 70 C) at acidic and neutral pH increases the aromatic surface hydrophobicity of the proteins. Furthermore, hydrophilic groups combined with water molecules and were buried inside the heated aggregation. The diminishing surface hydrophilic groups also increased the chance of ANS binding with aromatic hydrophobicity. Therefore, the combined effect of intense shear during superfine grinding and heat during microparticulation transformed the protein conformations. This phenomenon exposed the aromatic hydrophobic amino acids. 3.3. Steady-state fluorescence spectra Fig. 2A suggests that the maximum emission wavelength of WPC occurred at 333 nm. The maximum emission wavelengths of sWPC-4h and sWPC-8h shifted to longer wavelengths at 334 nm. After microparticulation, the maximum emission wavelength shifted to longer wavelength at 356 nm, and MPP-4h and MPP-8h continued to redshift to 358 nm (Fig. 2B). Given its aromatic character, tryptophan is often (although not always) found completely or partially buried in the hydrophobic core of protein interiors, at the interface between two
Fig. 1. Particle size distributions of proteins (A: powers; B: MPP).
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Table 2 Protein surface hydrophobicity (PSH) of samples. The values that do not bear the same letter in the same row are significantly different (p < 0.05). Samples
WPC
MWP
sWPC-4h
MPP-4h
sWPC-8h
MPP-8h
PSH
99.8 ± 10.2f
231.5 ± 16.8c
151.0 ± 3.5e
320.2 ± 2.5b
175.7 ± 7.2d
561.2 ± 13.3a
Fig. 2. Fluorescence emission spectrum of tryptophan.
protein domains or subdomains, or at the subunit interface in oligomeric protein systems. Disruption of the tertiary or quaternary structure of the protein further exposed the side chains to the solvent. In general, the exposure of the hydrophobic group can affect the ground- and excited-state energy levels of the chromophores, reduce the energy of the excited state, and cause the redshift of the emission spectrum (increased lambda Max) (Chan, Pinotsi, Kaminski Schierle, & Kaminski, 2014). In the present study, the redshift of the maximum emission wavelength of superfine and microparticulated proteins indicates the exposure of the hydrophobic group. This finding is consistent with the increased PSH of the proteins after superfine grinding and microparticulation. 3.4. Microrheology Figs. S1 and S2 illustrate the MSD curves at different aging times for the emulsions containing 40% and 80% oil. The measurements were started after sample preparation. The curves were displaced to larger time scales and developed more curvature with increasing oleaginousness, indicating increased viscosity and storage modulus, respectively (Pinder, Swanson, Hebraud, & Hemar, 2006). As illustrated in Fig. S1A1, B1, and C1, no significant movement was observed. This result indicates that the emulsions stabilized. The MSD curves moved from long to short as the superfine grinding time was prolonged. This result indicates increased elasticity. The same trend was observed for MWP (Fig. S2A1), MPP4h (Fig. S2B1), and MPP-8h (Fig. S2C1) emulsions containing 40% oil. However, when the oil content was increased to 80%, the sample showed the typical characteristic of a purely viscous product. That is, the MSD linearly increased with decorrelation time as the particles became completely free to move in the samples [Fig. S1A2, B2, and C2]. The MSD curves initially moved from long to short displacement (indicating increased elasticity) and from short to long decorrelation time (indicating increased braud, viscosity), and then stabilized (Bellour, Skouri, Munch, & He 2002). This step monitors network flocculation and stabilization.
Fig. S2A2, B2, and C2 shows the typical shape for the MSD of a viscoelastic product (concentrated emulsion, polymer solution with particles). Within short observation times, the particles can freely move in the continuous phase. The particles were blocked by their neighbors (or by polymers), and the MSD reached a plateau. This result is characteristic of product elasticity. Lower plateau resulted in tighter network and stronger elasticity. As the time scales were prolonged, the scatterers escaped from the “cages” formed by neighboring particles or polymers, and the MSD grew as it would for a viscous fluid. This result is characteristic of macroscopic viscosity because it corresponds to the speed of particles in the sample. In summary, the MSD is the viscoelastic fingerprint of the analyzed product. Table 3 shows the solideliquid balance (SLB), which corresponds to the MSD slope at short decorrelation time and represents the domination status of the sample liquid or solid. SLB ¼ 0.5 is a critical value of the transformation from liquid domination to solid domination. According to Tisserand, Fleury, Brunel, Bru, and Meunier (2012), the SLBs of WPC, sWPC-4h, and sWPC-8h emulsions containing 40% oil are greater than 0.5 and less than 1. This finding indicates that the liquid dominates the dispersions. Nevertheless, the SLBs of other emulsions were all smaller than 0.5. This finding indicates that the solid dominates the dispersions (gel behavior). Overall, the SLBs of sWPC-4h, sWPC-8h, MWP, MPP-4h, and MPP-8h emulsions containing the same amount of oil were significantly lower than that of WPC emulsion. Lower elastic plateau resulted in stronger elasticity. The elasticity index (EI) corresponds to the inverse of the MSD plateau value. As shown in Table 3, the EIs of sWPC-4h, sWPC-8h, MWP, MPP-4h, and MPP-8h emulsions containing the same amount of oil were significantly higher than that of WPC emulsion. This finding indicates increased elasticity. The macroscopic viscosity index (MVI), which corresponds to the inversed MSD slope in linear scale, is linked to the well-known macroscopic viscosity in Pa s. As shown in Table 3, the MVIs of sWPC-4h, sWPC-8h, MWP, MPP-4h, and MPP-8h emulsions containing the same amount of oil were significantly lower than that of WPC emulsion. This result indicates decreased viscosity. According
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Table 3 Solid liquid balance (SLB), elasticity index (EI) and macroscopic viscosity index (MVI) of emulsions at 10 min. The values that do not bear the same letter in the same column are significantly different (p < 0.05). Samples 40%
80%
WPC MWP sWPC-4h MPP-4h sWPC-8h MPP-8h WPC MWP sWPC-4h MPP-4h sWPC-8h MPP-8h
EI (103, nm2)
SLB 0.87 0.44 0.81 0.43 0.68 0.40 0.49 0.44 0.47 0.42 0.40 0.38
± ± ± ± ± ± ± ± ± ± ± ±
a
0.03 0.01fg 0.02b 0.01fh 0.02c 0.01ijk 0.02d 0.01f 0.02 de 0.01fghijk 0.01i 0.01ijl
1.15 1.10 1.14 1.63 1.18 1.71 2.86 17.50 2.85 67.23 1.68 160.37
± ± ± ± ± ± ± ± ± ± ± ±
ij
0.05 0.03ijkl 0.05ijk 0.05fgh 0.06i 0.02f 0.06d 0.17c 0.11de 1.56b 0.01fg 9.69a
MVI (104, nm2 s) 0.17 144.33 0.18 18.74 0.41 6.49 123.00 671.77 199.07 444.30 9.92 280.27
± ± ± ± ± ± ± ± ± ± ± ±
0.06ijkl 10.53e 0.02ijk 1.65g 0.08ij 0.02hi 8.61f 14.02a 13.70d 24.03b 1.13h 13.52c
to Mason (2000), higher macroscopic viscosity means more time is needed by the particles to travel a distance. Therefore, they have a lower particle speed, which may result in enhanced emulsion stability. 3.5. Emulsion stability The TSI curves of emulsions containing 40% oil showed a significant increasing trend, except for MPP-8h emulsions (Fig. 3A). The TSI curves of MPP emulsions containing 80% oil and MPP-8h emulsions containing 40% oil were flat and low (Fig. 3B). This result indicates high stability (Durand, Franks, & Hosken, 2003). The high TSI values can be attributed to the precipitation of protein and oil on the bottom and top of the vial, respectively (Wisniewska et al., 2013). Stable emulsions were defined as those that showed no significant changes in the oil droplet size and/or had no creaming or oiling off after storage. Table 4 shows the sum TSI of the emulsions at 24 h; this parameter reflects the storage stability of the emulsions. The TSI of the emulsions decreased as the time of superfine grinding was prolonged and after microparticulation. A decrease in TSI corresponded to an increase in stability. Thus, the stability of protein emulsions containing 40% and 80% oil increased after superfine grinding and microparticulation treatment. Similar findings were demonstrated by Dissanayake and Vasiljevic (2009) who postulated that the partial unraveling of WPs allows a thermodynamically favorable conformation for adherence to the watereoil interface.
A decrease in TSI corresponded to increases in PSH, MVI, EI, and oil level. On the basis of these findings, the enhanced aromatic hydrophobicity, MVI, EI, and oil levels were proposed to be the main factors regulating the stability of the MPP-8h emulsions upon storage. The combined effects of the first two factors predominantly affected the stability of the emulsions against creaming. The larger number of exposed aromatic hydrophobic residues increased the affinity of the protein molecules toward the oilewater interface. This phenomenon enhanced the proteinelipid interaction and simultaneously shortened the time required to reduce the surface tension, thereby producing small droplets. Furthermore, the large surface hydrophobicity may enhance the interaction between adjacent protein molecules that adsorbed at the interface, thereby forming a rigid and thick protein membrane that provides a considerable steric stabilization to the emulsion droplets. The enhanced emulsification of heat-denatured soy proteins and WPC possibly results from the exposure of buried hydrophobic groups (Surh, Gu, Decker, & McClements, 2005). The enhanced MVI of the continuous phase allowed the formation of a weak gel matrix that entrapped the fat droplets within the network, whereas increased oil levels enhanced the interactions between the droplets and protein; as a result, the movement of the droplets was retarded (Rosa, Sala, Van Vliet, & Van De Velde, 2006). 3.6. Droplet size distributions of the emulsions Fig. 4 shows the droplet size distributions of the emulsions containing 40% and 80% oil at 0 h. All of the droplet sizes of WPC and MWP emulsions containing 40% oil presented normal distribution (unimodal distribution) within approximately 15 mm (Fig. 4A). The droplet size distribution of sWPC-4h, sWPC-8h, MPP4h, and MPP-8h emulsions containing 40% presented another peak at approximately 3 mm. In addition, the peak height at approximately 3 mm increased as the time of superfine grinding treatment was prolonged. This result indicates that the number of small droplets increased. Meanwhile, the vertex ordinate indistinctively shifted to a smaller droplet size, which means the diminution of the droplet size with superfine grinding treatment. The same tendency was observed for the droplet size distributions of emulsions containing 80% oil. The conclusions are in accordance with the tendency of the mean droplet size (D50) (Table 5). Therefore, D50 was selected to intuitively characterize the droplet sizes.
Fig. 3. Turbiscan stability index (TSI) of emulsions containing (A) 40% and (B) 80% oil.
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Table 4 The Turbiscan stability index (TSI) of emulsions at 24 h ðx±SDÞ. The values that do not bear the same letter in the same row are significantly different (p < 0.05). Oil levels (%)
TSI WPC
40% 80%
MWP a
15.23 ± 0.35 11.19 ± 0.67a
sWPC-4h ab
14.55 ± 0.51 2.87 ± 0.44e
MPP-4h abc
14.88 ± 0.41 9.75 ± 0.94ab
sWPC-8h abce
12.06 ± 1.23 6.35 ± 0.29cd
MPP-8h cd
13.58 ± 0.68 8.53 ± 0.75bc
6.24 ± 1.26f 1.17 ± 0.02ef
Fig. 4. Droplet size distributions of the emulsions (A: emulsions containing 40% oil; B: emulsions containing 80% oil).
Table 5 Median particle size (D50) of the emulsion droplets. The values that do not bear the same letter in the same row are significantly different (p < 0.05). Oil levels (%)
40% 80%
D50 (mm) WPC
MWP
sWPC-4h
MPP-4h
sWPC-8h
MPP-8h
16.2 ± 0.1a 13.5 ± 1.0a
11.1 ± 1.5bcde 9.3 ± 1.2bc
13.4 ± 0.6ab 9.5 ± 0.5ef
11.3 ± 0.3bd 10.0 ± 0.2b
12.1 ± 3.0bcd 9.2 ± 0.5bcd
7.9 ± 1.0def 8.5 ± 0.3bcde
No significant difference in D50 was observed between WPC and sWPC-4h emulsions containing 40% and 80% oil. However, the D50 of sWPC-8h emulsions containing 40% and 80% oil was significantly lower (p < 0.05) than that of the emulsions containing the same levels of oil. The observed behavior can be ascribed to the increased adsorbance efficiency of sWPC-8h onto the oilewater interface. In general, the fat droplet size decreased with increasing oil levels regardless of protein type. This result may be attributed to the fact that the emulsions were constructed by a loose network of spherical droplets that progressed to form a compact matrix as the oil level was increased to 80%. The findings agreed with the observation by Hemar and Horne (2000) for caseinate-stabilized
emulsions in which the spherical, close packing limit was shown to fall around 70% oil. The authors reported hexagonal and pentagonal structures in emulsions with a highly dispersed phase volume. Therefore, the high emulsibility of the sWPC- and MPPstabilized emulsions can be advantageously exploited in the manufacture of spread-like reduced-fat and gel-like high-fat foods. 4. Conclusions Superfine grinding and microparticulation were employed to produce new WPC ingredients, namely, sWPC and MPP. The redshift of the maximum emission wavelength (from 333 nm to
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334 nm, 356 nm, and 358 nm) and the increase in PSH indicated an increase the hydrophobicity of WPC and changes in the microenvironment of the fluorophore inside the protein molecule. The decrease in SLB and the increases in EI and MVI were attributed to the increased ratio of oil phase, superfine grinding treatment, and microparticulation. This result indicates that the status of the emulsions changed from liquid to viscoelastic behavior. The TSI of the emulsions at 24 h directly reflected the higher emulsibility of sWPC, and the use of MPP as the continuous phase resulted in concentrated emulsions containing 40% and 80% oil. These emulsions remained stable even after prolonged storage of 24 h at 25 C. These findings imply that enhanced PSH, MVI, EI, and higher oil level are the main factors regulating the stability of stabilized emulsions upon storage. The results of this study confirmed our hypothesis that reactive superfine grinding and microparticulation render WPC into an ingredient with excellent gelling and emulsifying properties. This finding may widen the applications of sWPC and MPP in food emulsions. sWPC and MPP with an excellent emulsion-stabilizing property can be utilized in both reduced-fat and high-fat food applications requiring high stability. Acknowledgments The authors acknowledge the financial support from the National High Technology Research and Development Program of China (863 Program) (Grant no. 2013AA102204). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.foodhyd.2015.05.027. References Alizadeh-Pasdar, N., & Li-Chan, E. C. (2000). Comparison of protein surface hydrophobicity measured at various pH values using three different fluorescent probes. Journal of Agricultural and Food Chemistry, 48(2), 328e334. braud, P. (2002). Brownian motion of Bellour, M., Skouri, M., Munch, J.-P., & He particles embedded in a solution of giant micelles. The European Physical Journal E: Soft Matter and Biological Physics, 8(4), 431e436. Chan, F. T., Pinotsi, D., Kaminski Schierle, G., & Kaminski, C. F. (2014). Structurespecific intrinsic fluorescence of protein amyloids used to study their kinetics of aggregation. Academic Press. Chen, H., Weiss, J., & Shahidi, F. (2006). Nanotechnology in nutraceuticals and functional foods. Food Technology, 60(3), 30e36. Chen, Y. Q., Chou, P.l., Cheng, C. Y., Chiang, C. C., Wei, M. T., Chuang, C. T., et al. (2012). Microrheology of human synovial fluid of arthritis patients studied by diffusing wave spectroscopy. Journal of Biophotonics, 5(10), 777e784. Dissanayake, M., & Vasiljevic, T. (2009). Functional properties of whey proteins affected by heat treatment and hydrodynamic high-pressure shearing. Journal of Dairy Science, 92(4), 1387e1397.
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