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Structure and dilatational rheological behavior of heat-treated lotus (Nelumbo nucifera Gaertn.) seed protein
LWT - Food Science and Technology 116 (2019) 108579
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Structure and dilatational rheological behavior of heat-treated lotus (Nelumbo nucifera Gaertn.) seed protein
T
Xiangze Jiaa,b, Jianyi Wanga,b, Xu Lua,b, Bingxin Zhenga, Baodong Zhenga,b, Zebin Guoa,b,∗ a b
College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian, PR China China-Ireland International Cooperation Centre for Food Material Science and Structure Design, Fujian Agriculture and Forestry University, Fuzhou, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Heat treatment Interfacial properties Hydrophobicity Surface potential Surface pressure
The influences of heat treatment (60, 80 and 100 °C, 30min) on the structure and interfacial properties (adsorption at the oil−water interface and dilatational rheology of interfacial layers) of lotus seed protein (LSP) were evaluated. Heat treatment induced an increase in average diameter and surface hydrophobicity due to partial unfolding and aggregation of LSP. Specifically, the moderate heat treatment (60 and 80 °C) could significantly increase the net surface potential of LSP, while heating at 100 °C had no effect. Additionally, the lower surface pressure at long-term adsorption and similar dynamic interfacial rheology were observed as compared to native LSP. In contrast, heat treatment led to a higher diffusion and penetration rate of protein, as observed by a faster increase of surface pressure. Nevertheless, the viscoelastic properties of the adsorbed layer of LSP after heat treatment decreased and when the interfacial pressure was greater than 18 mN/m, the interfacial modulus showed a downward trend. These findings increase our understanding of the structural dependence of protein interface behavior and the application of heat-treated proteins in emulsion preparation.
1. Introduction
In general, heat treatment induces de-folding and aggregation of the protein structure, resulting in the protein exhibiting a different surface hydrophobicity and size from the native state (Kitabatake, Hatta, & Doi, 2014; Liu & Tang, 2013). For oil/water emulsion systems, these changes affect the diffusion, permeation and rearrangement of proteins from bulk to interface, resulting in different viscoelastic properties of the interface adsorption layer (Wang et al., 2012). On the other hand, the interfacial properties of proteins can provide useful insights into their ability to stabilize emulsions and foams. Therefore, in order to know more about the relationship between heat-denatured protein structure and functional properties, more attention has been paid to the adsorption behavior of proteins at the interface (Liu and Tang 2014, 2016; Wang et al., 2012). However, the interfacial properties of proteins are also closely related to their own structure. For example, heat treatment can enhance the surface activity of whey protein isolate (Rodríguez Patino, Rodríguez Niño, & Sánchez, 1999), lactalbumin (Wijesinha-Bettoni et al., 2007), lactoglobulin (Kim, Cornec, & Narsimhan, 2005) and soy protein (Wang et al., 2012) and the viscoelasticity of the adsorbed layer, whereas ovalbumin showed a lower shear elastic constant after heating at 80 °C for 40 min (Croguennec, Renault, Beaufils, Dubois, & Pezennec, 2007). Therefore, based on these considerations, this study examined the effects of different degrees of heat treatment (60, 80 and 100 °C) on the
Lotus seed protein (LSP) is the second most abundant ingredient in lotus seeds, its content is about 16%–17% (w/w) (Sridhar & Bhat, 2007, Zeng, Cai, Cai, Wang, & Li, 2013; Zhang et al., 2015). The composition of lotus protein mainly includes albumin (41.58%), globulin (26.58%), gluten (18.0%) and gliadin (6.0%) (Zhang et al., 2015). Importantly, lotus protein is rich in essential amino acids, with a nutritional value close to that of soy protein (Pan, Zeng, Alain, & Feng, 2016). Therefore, the research interests of lotus protein as a functional food ingredient have been significantly increasing in recent years (Zeng et al., 2013). Generally, the application of proteins in the food industry is mainly based on their excellent interface (emulsifying and foaming) and gel properties (Wang et al., 2012). However, as lotus protein is a relatively new type of food protein source, details relating to its functional properties are still very limited, which severely limits its widespread use in the food industry. On the other hand, heat treatment is a widespread unit operation in the food industry. For example, most foods require heat sterilization to increase their shelf life. This will inevitably affect the structure and characteristics of proteins in the food system. Therefore, clarifying the effect of heat treatment on the structure and properties of proteins can provide guidance for their appropriate application in the food industry. ∗
Corresponding author. College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian, 350002, PR China. E-mail address: [email protected] (Z. Guo).
https://doi.org/10.1016/j.lwt.2019.108579 Received 21 May 2019; Received in revised form 27 August 2019; Accepted 30 August 2019 Available online 30 August 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
LWT - Food Science and Technology 116 (2019) 108579
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(PDI) of each sample were measured with the same instrument. The zeta potential and particle size were automatically determined by the instrument using the Henry equation and Mie theory, respectively. The refractive index of solvents was 1.33 and the refractive index of nanoparticles was 1.50. All tests were conducted in triplicate at 25 °C.
LSP structure and the automated drop tensiometer was used to study the LSP adsorption and rearrangement characteristics at the oil-water interface both before and after heat treatment. The focus of this work was to establish the link between the structural change of the LSP and its interfacial rheological properties, thereby providing guidance for the application of LSP, a new type of protein resource, for the development of functional food ingredients (e.g., emulsifier).
2.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE)
2. Materials and methods
Lotus seed powder was provided by Green Field Fujian Food Co. Ltd (Fujian, China). 8-anilinonaphthalene-sulfonic acid (ANS) obtained from Sigma-Aldrich Company (St. Louis, MO, USA). Other reagents were purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Medium chain triglyceride (MCT) was purchased from Sigma Co. (St. Louis, MO) without further purification. All of the other reagents were of analytical grade.
SDS-PAGE was conducted according to the method of (Xiong et al., 2016). In brief, a 12% (w/v) acrylamide separating gel and a 5% (w/v) staking gel containing 10% SDS was performed using electrophoresis (Liuyi, China). A LSP dispersion (0.2 mM) was prepared in sample buffer (10 Mm Tris-HCl, 10% (w/w) glycerinum, 0.02% (w/w) bromophenol blue, 2% (w/w) SDS and 5% (v/v) β-mercaptoethanol, pH 8.0). The protein solution was heated for 3 min at 100 °C and centrifuged at 1500 g for 10 min. Gels were stained with Coomassie Brilliant Blue R-250 and destained with methanol: acetic acid: water (50:10:40) (v/v/v) acetic acid in water.
2.2. Fabrication and heat treatment of LSP
2.6. Measurement of circular dichroism (CD) spectrum
Fabrication of LSP: The LSP was extracted as previously described by Pan et al. (2016). Briefly, the lotus seed powder was mixed with 100 mL of 0.10 mol/L NaCl and constant magnetic stirring (100 rpm) was applied for 1 h at 40 °C. The mixture was then centrifuged for 20 min at 2800 g using a refrigerated centrifuge (Avanti J-26 XP centrifuge, Beckman Coulter, Fullerton, CA, USA). Afterwards, the supernatant was adjusted to pH 4.2 by adding 0.1 mol/L HCl solution (acid precipitation), and then re-centrifuged (4800 g for 25 min). The precipitate obtained was taken to be the crude LSP product. The crude product was washed with deionized water (pH 4.2) four times and subsequently freeze-dried to give the LSP powder. The LSP was mainly composed of globulin, and its protein content was determined to be greater than 90% by Kjeldahl method. Heat treatment of LSP: LSP freeze-dried powder was dissolved in deionized water at 1% (w/v) total solids and the solution was slowly stirred for 4 h at 25 °C and stored overnight at 4 °C to ensure complete hydration. In addition, sodium azide (0.02% w/v) was added to the LSP solution to inhibit bacteria growth. Samples were subjected to different heat treatments (0, 60, 80, 100 °C) for 30 min at pH 7.0 without salt ion. After heat treatment, all samples were quickly cooled in an ice-water bath and then stored in a 4 °C environment until analyzed.
The LSP solution (1% w/v), both before and after heat treatment, was diluted in 10 mmol/L phosphate buffer, pH 7.0 to obtain the 0.02% (w/v) test sample. The CD spectrum was performed using a spectropolarimeter (Jasco 810, Jasco Corp., Tokyo, Japan) in the range 190–240 nm at 25 °C. For this test, a quartz cuvette was used with an optical path of 0.1 cm. The spectral resolution was 0.5 nm, the scan speed was 100 nm/min and the response time was 0.25 s with a bandwidth of 1 nm. Three scans were accumulated and averaged.
2.1. Materials
2.7. Measurement of fluorescence spectrum The test protein solution 0.02% (w/v) was prepared as described in the CD experiment. The intrinsic fluorescence spectroscopy of LSP was performed using a Luminescence Spectrometer (F-4600, Japan) at 25 °C. A quartz cuvette with an optical path of 1 cm was used. The excitation wavelength was 290 nm and the emission spectra ranged from 300 to 460 nm. The excitation and emission slit widths were 2.5 nm and a scan speed of 1200 nm/min was used. 2.8. Dynamic interface properties measurement The interfacial surface pressure (π) and adsorption kinetics of LSP both before and after heat treatment at the oil/water interface were performed using an automated drop tensiometer (Tracker-H, Teclis, France) at 25 °C (Xiong et al., 2018). The LSP solution (1%, w/v) and oil phase (medium chain fatty acid, MCT) were placed in the cuvette and syringe, respectively. The test used the hanging drop method. Before testing, dispersions and oil were allowed to stand for at least 1 h to reach 25 °C. The temperature of the system was maintained constant by circulating water from a thermostat. The oil droplet volume during the test was 10 μL. The oil/water π (mN/m) was calculated with equation (1):
2.3. Measurement of surface hydrophobicity (H0) The surface hydrophobicity of LSP was determined by a fluorescence spectrum assay using 8-anilino-1-naphthalenesulfonic acid (ANSA) as a fluorescent probe (Delahaije, Wierenga, Nieuwenhuijzen, Giuseppin, & Gruppen, 2013). The LSP and ANSA were dissolved in phosphate buffered saline (PBS, pH 7.0, 10 mmol/L) to obtain a 1 mg/ mL and 2.4 mmol/L solution, respectively. Subsequently, aliquots of 50 μL ANSA solutions were added into 5 mL LSP solution for analysis. The excite wavelength was set at 385 nm and the emission wavelength range was 400–650 nm using a fluorescence spectrophotometer (Shimadzu RF-5310PC, Japan). The emission and excitation slits were set at 5 nm and the measurements were performed at 25 °C. The area of the fluorescence spectrum was corrected with the area of the buffer and the relative exposed hydrophobicity was expressed as the area of the sample.
π (mN/m) = γ0-γ
(1)
Where γ0 (mN/m) and γ (mN/m) is the interfacial tension of pure oildistilled water (26 mN/m) and LSP solutions, respectively. The surface tension was determined by drop shape analysis (Benjamins, Cagna, & Lucassen-Reynders, 1996). All measurements were performed for up to 3 h. Generally, there is a rate-determining step, showing relatively low interfacial surface pressures during the first step (Camino, Pérez, Sanchez, Patino, & Pilosof, 2009). The change in interfacial π with adsorption time (t) can be correlated by a modified form of the Ward and Tordai equation:
2.4. Measurement of particle size and zeta potential The zeta potentials of fresh, undiluted samples were determined using a microelectrophoresis instrument (ZS Zetasizer Nano, Malvern Instrument Ltd., UK). The average particle size and polydispersity index 2
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π = 2C0KBT(Dt/3.14)1/2
2012). Additionally, Fig. 1b shows the far-UV CD spectra of untreated and heated LSP. From the intensity of the CD spectra, some changes can be observed in the secondary structure of LSP. The spectra were analyzed using Yang's equation and the proportions of α-helix, β-sheet, β-turn and random coil was ~0%, 82%, 0% and 18%, respectively. These values conflict with those previously reported for native LSP (Zeng, Cai, Cai, Wang, & Li, 2011). This phenomenon could be attributed to the different LSP composition and characterization means used by Zeng et al. (2011) to investigate the secondary structure of LSP. However, the differences in these values between samples was < 1%. It was deduced from these spectra that the secondary structure was not significantly affected by the heat treatment. The effects of heating on the tertiary structure of LSP were examined by the intrinsic fluorescence, which can be reflected by the micro-environmental changes around tryptophan residues of LSP. A maximum fluorescence intensity was observed at 348 nm for both native and heated LSP (Fig. 1c). The fluorescence intensity decreased with an increase in heat treatment temperature from 0 to 100 °C. This variation in fluorescence intensity may be ascribed to the difference in energy transfer efficiency between tryptophan and tyrosine, as well as the chromophores of LSP becoming exposed to solvents due to heat treatment (Xiong et al., 2016). Generally, tyrosine, tryptophan and phenylalanine residues in proteins, particularly tryptophan residues, will be fluorescently polarized in a manner that is highly dependent on protein folding and serve as a tertiary structure for sensitivity monitors (Xiong et al., 2016). Therefore, this result shows that the tertiary structure of LSP had been changed and partly unfolded due to heat treatment, especially for the 100 °C (30 min) heating.
(2)
Where C0 is the concentration in the continuous phase, KB is the Boltzmann constant, T is the absolute temperature and D is the diffusion coefficient. If the adsorption process is controlled by the protein diffusion, a plot of π versus t1/2 will then be linear and the slope of this plot will be the diffusion rate (Kdiff). To obtain interface dilatational parameters, the dynamic interface viscoelasticity of LSP solutions at the oil-water interface were also investigated. The sinusoidal interfacial compression and expansion were performed by changing the drop volume at 10% of the deformation amplitude within the linear regime, the oscillation frequency was 0.1 Hz, the droplet volume was 10 μL. The experiment was started after an equilibration period of 60 s. The number of active and blank was 5 cycles during the experiments. Details of this experiment were described by Perez, Carrara, Sánchez, Santiago, and Patino (2009). The interface dilatational modulus (E) derived from the change in interfacial tension (γ), resulting from a small change in surface area (A), can be described by equation (3): E = dγ/(dA/A) = − dπ/dln (A) = Ed + iEv
(3)
The E is a complex quantity and is composed of real and imaginary parts. The real part of E or storage component is interface dilatational elasticity (Ed). The imaginary part of the E or the loss component is the interface dilatational viscosity (Ev) (Beverung, Radke, & Blanch, 1999). 2.9. Statistical analysis All measurements were carried out in triplicate unless otherwise stated. One-way analysis of variance (ANOVA) with a 95% confidence interval was used to assess the significance of the results obtained. Statistical analysis was performed using SPSS software version 19.0.
3.2. Effects of heat treatment on the size, zeta potential and surface hydrophobicity of LSP
3. Results and discussion
The influence of heating on the average diameter distribution of LSP was conducted by dynamic light scattering (DLS) and the results obtained are presented in Fig. 2a. As expected, all heat treatments resulted in a significant increase in protein mean diameter compared to native LSP, which was due to partial unfolding and aggregation of the protein molecules during heating. Meanwhile, the size of LSP aggregates tended to increase as the heat treatment temperature increased. It is noteworthy that the dimensional distribution curve of the LSP after heating at 80 °C for 30 min showed two peaks, which should be related to the degree of aggregation of the protein under different heating conditions (Liu & Tang, 2014; Wang et al., 2012). This was confirmed by the change of zeta potential and surface hydrophobicity of LSP after heat
3.1. Effects of heat treatment on the structure of LSP The impacts of heat treatment on the primary structure of LSP were evaluated by SDS-PAGE. A comparison of the electrophoretic patterns of the proteins revealed no significant difference between the treated and untreated LSP in terms of their molecular weights, indicating that no significant changes occurred in the primary structure of the protein (Fig. 1a). This suggests that heat treatment does not affect the individual subunits of LSP. A similar phenomenon was also observed in the soybean protein isolate system after heat treatment (Wang et al.,
Fig. 1. Structural characterization of LSP fractions. (a) SDS-PAGE electrophoretic profile of untreated (A) and heated (B–D) LSP. M is the marker. (b) Far-UV CD spectra of untreated (A) and heated (B–D) LSP. (c) Emission fluorescence spectra of untreated (A) and heated (B–D) LSP. The black, red, blue and green lines in Figure b and c are unheated, heat treated at 60 °C, 80 °C, and 100 °C for 30 min, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 3
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Fig. 2. Effect of heating on the particle size (a), zeta potential (b) and surface hydrophobicity (c) of untreated (A) and heated (B–E) LSP. The black, red, blue and green lines in Figure a are unheated, heat treated at 60 °C, 80 °C, and 100 °C for 30 min, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
(Fig. 3A). In addition, the rates of penetration and rearrangement of adsorbed protein molecules at the oil-water interface can be determined by the first-order equation:
treatment (Fig. 2b, c). Compared with natural LSP, the net surface potential of LSP significantly increased after heating (P < 0.05), which is related to the charged amino acid residues on the surface of the protein, except in samples heated at 100 °C for 30 min (P > 0.05). This change is likely related to the charged amino acid residues on the surface of the protein (Delahaije, Gruppen, Giuseppin, & Wierenga, 2014). In addition, the surface hydrophobicity of the LSP was also significantly enhanced after heat treatment (P < 0.05), meaning that hydrophobic residues within the protein molecule became exposed. These results further demonstrate that the degree of unfolding and aggregation of LSP structures are various under different temperature treatments.
ln(πf-πt)/ (πf-π0) = -kit
(4)
Where πf, π0 and πt are the interfacial pressures at the final adsorption time, at the initial time and at any time of each stage, respectively and ki is the first-order rate constant. The application of Eq. (4) to the adsorption of the LSP both before and after heating at the interface is presented in Fig. 3B. Clearly, there were two linear regions in these plots. Generally, the first slope is usually regarded as the rate constant of penetration (KP) and the second slope as the rate constant of molecular reorganization (Kr) (Perez et al., 2009). The rate constants of LSP molecular diffusion (Kdiff), penetration (Kp), reorganization and the equilibrium interfacial pressure (π7200) at the oil-in-water interface are summarized in Table 1. The Kdiff, Kp and π7200 of the LSP without heating was lower than the samples after heat treatment, which might be mainly due to the enhancement of surface hydrophobicity. In general, the interface properties of protein were greatly related to the surface charge, surface hydrophobicity, molecular flexibility and particle size (Morales, Martínez, Ruiz-Henestrosa, & Pilosof, 2015; Wierenga, Meinders, Egmond, Voragen, & de Jongh, 2003; Wierenga, Meinders, Egmond, Voragen, & de Jongh, 2005). The
3.3. Interfacial adsorption and dilatational rheological properties 3.3.1. Adsorption kinetics and structural rearrangements at the oil-in-water interface The interface adsorption behavior of protein molecules at the oil–water interface is mainly influenced by diffusion, conformational rearrange, and actual adsorption (penetration and unfolding) (Dickinson, 2011). Accordingly, the rate at which protein molecules diffuse from the bulk phase to the oil–water interface (Kdiff) can be determined by the linear slope of the interface pressure versus adsorption time curve
Fig. 3. (A) Time dependence of surface pressure (π) for LSP adsorbed layer at the oil-water interface. Kdiff represent diffusion rate. (B) Typical profile of the molecular penetration and rearrangement steps at the oil-water interface for LSP. Kp and Kr represent first-order rate constants of penetration and rearrangement, respectively. The black, red, blue and green dots in the figure are unheated, heat treated at 60 °C, 80 °C, and 100 °C for 30 min, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 4
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Table 1 The diffusion rate (Kdiff), constants of penetration and structural rearrangement at the interface (KP and KR), and surface pressure at the end of adsorption (7200 s, π7200) for LSP pH 7.0. Kdiff (mN/m/s1/2) (LR) A (Unheated) B (60 °C,30min) C (80 °C,30min) D (100 °C,30min)
0.19 0.23 0.25 0.26
± ± ± ±
0.03 0.01 0.02 0.02
(0.9533)b (0.9790)ab (0.9746)a (0.9829)a
Kp × 10−4 (LR) −3.12 −4.02 −4.05 −4.13
± ± ± ±
0.03 0.03 0.02 0.02
Kr × 10−3 (LR) (0.9870)c (0.9976)b (0.9741)b (0.9855)a
−3.33 −3.71 −3.30 −3.18
± ± ± ±
0.03 0.05 0.05 0.03
Π7200 (mN/m) (0.8970) b (0.8853) a (0.9415)b (0.9152)c
17.71 19.60 20.05 20.13
± ± ± ±
0.06c 0.10ab 0.06a 0.06a
LR is an abbreviation for linear regression coefficients. Different letters in the same column indicate significant differences (P < 0.05).
interfacial adsorption layer, thereby exhibiting a lower viscoelastic property (E value). On the other hand, as more LSP molecules adsorb to the interfacial layer, they may cause the already adsorbed protein molecules to dissociate from the interface and contribute to the decreasing trend in the E value. Furthermore, the dynamic dilatational elastic moduli (Ed) of interfacial layers of all samples are presented in Fig. 4B. Clearly, the Ed values gradually increased due to the adsorption of protein at the interface and almost showed the same trend as E. After adsorption for 120 min, the Ed values were close to the surface dilatational modulus (E values) and the dilatational viscosity values were low (data not shown). This result suggested that the interface absorbed layer mainly exhibited the elastic behavior.
increase of protein particle size and surface net potential could play a negative role for the diffusion and adsorption rate of protein in oilwater and air-water interfaces. In all cases, the interfacial surface pressure increased with adsorption time (Fig. 3A), which could be ascribed to the LSP adsorption at the interface (Perez et al., 2009; Wan, Wang, Wang, Yuan, & Yang, 2014). On the other hand, Kr for the adsorbed LSP was considerably higher than Kp, suggesting the importance of the structural rearrangement of adsorbed LSP at the interface to film formation rather than their penetration. 3.3.2. Dilatational rheological properties at the oil-in-water interface The stability of the protein emulsion can be predicted by studyingthe viscoelastic properties of the protein-adsorbed layer at the oil–water interface (Murray, 2002). Therefore, the surface E as a function of interfacial π in the surface layer for the adsorption of LSP before and after heating is presented in Fig. 4A. The E-π curve and its slope reflect the equilibrium state of the adsorbed protein at the interface. If the increase of E is only related to the adsorption amount of the surfactant on the interface, the E data of all the systems should be approximately the same in the E-π curve. It can be observed from Fig. 4A that E increased with the increase of the π value for all samples, indicating that there was interaction between the adsorbed protein molecular residues which were gradually increasing. The slopes of the E-π curves were greater than 1, which demonstrates a non-ideal adsorption state, suggesting that there was a strong interaction between the adsorbed protein molecules. Similar results were also observed in other heated protein systems (Liu & Tang, 2014; Wang et al., 2012). However, when the π was greater than 18 mN/m, the E value exhibited a decreasing trend and the heat-treated samples had lower E values than the native LSP. This result can be explained as the LSP size and the surface electrostatically increased due to the heat treatment, resulting in a decrease in the interaction between the protein molecules of the
4. Conclusion Heat treatment induced partial de-folding and aggregation of the LSP structure, exhibiting greater average diameter and surface hydrophobicity. However, heating showed multiple effects on the surface potential of LSP. Moderate heat treatment (60 and 80 °C) can significantly increase the surface net potential of LSP, while heating at 100 °C for 30 min has no significant effect. In addition, the diffusion and permeation rate of LSP at the oil-water interface were increased after heat treatment and exhibited higher surface pressure values. However, the viscoelastic properties of the interface adsorption layer have not been enhanced and the interfacial rheological behavior dominated by the elastic modulus has been demonstrated. It is speculated that the above findings may be related to the change of LSP structure caused by heat treatment.
Fig. 4. (A) Surface dilatational modulus (E) as a function of surface pressure (π) for LSPs at the oil−water interface. (B) Time-dependent dilatational elasticity (Ed) for LSPs adsorbed layers at the oil-water interface. The black, red, blue and green dots in the figure are unheated, heat treated at 60 °C, 80 °C, and 100 °C for 30 min, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 5
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Declaration of interest statement
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We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled “Structure and dilatational rheological behavior of heat-treated lotus (Nelumbo nucifera Gaertn.) seed protein”. Acknowledgments The authors gratefully acknowledge the financial support from the International Science and Technology Cooperation and Exchange Project of Fujian Agriculture and Forestry University (KXGH17001), the Science and Technology Major Project in Fujian Province (2018NZ0003-1), and the FAFU Funds for Distinguished Young Scientists (xjq201618). References Benjamins, J., Cagna, A., & Lucassen-Reynders, E. H. (1996). Viscoelastic properties of triacylglycerol/water interfaces covered by proteins. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 114, 245–254. Beverung, C. J., Radke, C. J., & Blanch, H. W. (1999). Protein adsorption at the oil/water interface: Characterization of adsorption kinetics by dynamic interfacial tension measurements. Biophysical Chemistry, 81(1), 59–80. Camino, N. A., Pérez, O. E., Sanchez, C. C., Patino, J. M. R., & Pilosof, A. M. (2009). Hydroxypropylmethylcellulose surface activity at equilibrium and adsorption dynamics at the air–water and oil–water interfaces. Food Hydrocolloids, 23(8), 2359–2368. Croguennec, T., Renault, A., Beaufils, S., Dubois, J. J., & Pezennec, S. (2007). Interfacial properties of heat-treated ovalbumin. Journal of Colloid and Interface Science, 315(2), 627–636. Delahaije, R. J., Gruppen, H., Giuseppin, M. L., & Wierenga, P. A. (2014). Quantitative description of the parameters affecting the adsorption behaviour of globular proteins. Colloids and Surfaces B: Biointerfaces, 123, 199–206. Delahaije, R. J. B. M., Wierenga, P. A., Nieuwenhuijzen, N. H. V., Giuseppin, M. L. F., & Gruppen, H. (2013). Protein concentration and protein-exposed hydrophobicity as dominant parameters determining the flocculation of protein-stabilized oil-in-water emulsions. Langmuir, 29(37), 11567–11574. Dickinson, E. (2011). Mixed biopolymers at interfaces: Competitive adsorption and multilayer structures. Food Hydrocolloids, 25(8), 1966–1983. Kim, D. A., Cornec, M., & Narsimhan, G. (2005). Effect of thermal treatment on interfacial properties of β-lactoglobulin. Journal of Colloid and Interface Science, 285(1), 100–109. Kitabatake, N., Hatta, H., & Doi, E. (2014). Heat-induced and transparent gel prepared from hen egg ovalbumin in the presence of salt by a two-step heating method. Agricultural & Biological Chemistry, 51(3), 771–778. Liu, F., & Tang, C. H. (2013). Soy protein nanoparticle aggregates as pickering stabilizers for oil-in-water emulsions. Journal of Agricultural and Food Chemistry, 61(37),
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Structure and dilatational rheological behavior of heat-treated lotus (Nelumbo nucifera Gaertn.) seed protein
T
Xiangze Jiaa,b, Jianyi Wanga,b, Xu Lua,b, Bingxin Zhenga, Baodong Zhenga,b, Zebin Guoa,b,∗ a b
College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian, PR China China-Ireland International Cooperation Centre for Food Material Science and Structure Design, Fujian Agriculture and Forestry University, Fuzhou, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Heat treatment Interfacial properties Hydrophobicity Surface potential Surface pressure
The influences of heat treatment (60, 80 and 100 °C, 30min) on the structure and interfacial properties (adsorption at the oil−water interface and dilatational rheology of interfacial layers) of lotus seed protein (LSP) were evaluated. Heat treatment induced an increase in average diameter and surface hydrophobicity due to partial unfolding and aggregation of LSP. Specifically, the moderate heat treatment (60 and 80 °C) could significantly increase the net surface potential of LSP, while heating at 100 °C had no effect. Additionally, the lower surface pressure at long-term adsorption and similar dynamic interfacial rheology were observed as compared to native LSP. In contrast, heat treatment led to a higher diffusion and penetration rate of protein, as observed by a faster increase of surface pressure. Nevertheless, the viscoelastic properties of the adsorbed layer of LSP after heat treatment decreased and when the interfacial pressure was greater than 18 mN/m, the interfacial modulus showed a downward trend. These findings increase our understanding of the structural dependence of protein interface behavior and the application of heat-treated proteins in emulsion preparation.
1. Introduction
In general, heat treatment induces de-folding and aggregation of the protein structure, resulting in the protein exhibiting a different surface hydrophobicity and size from the native state (Kitabatake, Hatta, & Doi, 2014; Liu & Tang, 2013). For oil/water emulsion systems, these changes affect the diffusion, permeation and rearrangement of proteins from bulk to interface, resulting in different viscoelastic properties of the interface adsorption layer (Wang et al., 2012). On the other hand, the interfacial properties of proteins can provide useful insights into their ability to stabilize emulsions and foams. Therefore, in order to know more about the relationship between heat-denatured protein structure and functional properties, more attention has been paid to the adsorption behavior of proteins at the interface (Liu and Tang 2014, 2016; Wang et al., 2012). However, the interfacial properties of proteins are also closely related to their own structure. For example, heat treatment can enhance the surface activity of whey protein isolate (Rodríguez Patino, Rodríguez Niño, & Sánchez, 1999), lactalbumin (Wijesinha-Bettoni et al., 2007), lactoglobulin (Kim, Cornec, & Narsimhan, 2005) and soy protein (Wang et al., 2012) and the viscoelasticity of the adsorbed layer, whereas ovalbumin showed a lower shear elastic constant after heating at 80 °C for 40 min (Croguennec, Renault, Beaufils, Dubois, & Pezennec, 2007). Therefore, based on these considerations, this study examined the effects of different degrees of heat treatment (60, 80 and 100 °C) on the
Lotus seed protein (LSP) is the second most abundant ingredient in lotus seeds, its content is about 16%–17% (w/w) (Sridhar & Bhat, 2007, Zeng, Cai, Cai, Wang, & Li, 2013; Zhang et al., 2015). The composition of lotus protein mainly includes albumin (41.58%), globulin (26.58%), gluten (18.0%) and gliadin (6.0%) (Zhang et al., 2015). Importantly, lotus protein is rich in essential amino acids, with a nutritional value close to that of soy protein (Pan, Zeng, Alain, & Feng, 2016). Therefore, the research interests of lotus protein as a functional food ingredient have been significantly increasing in recent years (Zeng et al., 2013). Generally, the application of proteins in the food industry is mainly based on their excellent interface (emulsifying and foaming) and gel properties (Wang et al., 2012). However, as lotus protein is a relatively new type of food protein source, details relating to its functional properties are still very limited, which severely limits its widespread use in the food industry. On the other hand, heat treatment is a widespread unit operation in the food industry. For example, most foods require heat sterilization to increase their shelf life. This will inevitably affect the structure and characteristics of proteins in the food system. Therefore, clarifying the effect of heat treatment on the structure and properties of proteins can provide guidance for their appropriate application in the food industry. ∗
Corresponding author. College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian, 350002, PR China. E-mail address: [email protected] (Z. Guo).
https://doi.org/10.1016/j.lwt.2019.108579 Received 21 May 2019; Received in revised form 27 August 2019; Accepted 30 August 2019 Available online 30 August 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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(PDI) of each sample were measured with the same instrument. The zeta potential and particle size were automatically determined by the instrument using the Henry equation and Mie theory, respectively. The refractive index of solvents was 1.33 and the refractive index of nanoparticles was 1.50. All tests were conducted in triplicate at 25 °C.
LSP structure and the automated drop tensiometer was used to study the LSP adsorption and rearrangement characteristics at the oil-water interface both before and after heat treatment. The focus of this work was to establish the link between the structural change of the LSP and its interfacial rheological properties, thereby providing guidance for the application of LSP, a new type of protein resource, for the development of functional food ingredients (e.g., emulsifier).
2.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE)
2. Materials and methods
Lotus seed powder was provided by Green Field Fujian Food Co. Ltd (Fujian, China). 8-anilinonaphthalene-sulfonic acid (ANS) obtained from Sigma-Aldrich Company (St. Louis, MO, USA). Other reagents were purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Medium chain triglyceride (MCT) was purchased from Sigma Co. (St. Louis, MO) without further purification. All of the other reagents were of analytical grade.
SDS-PAGE was conducted according to the method of (Xiong et al., 2016). In brief, a 12% (w/v) acrylamide separating gel and a 5% (w/v) staking gel containing 10% SDS was performed using electrophoresis (Liuyi, China). A LSP dispersion (0.2 mM) was prepared in sample buffer (10 Mm Tris-HCl, 10% (w/w) glycerinum, 0.02% (w/w) bromophenol blue, 2% (w/w) SDS and 5% (v/v) β-mercaptoethanol, pH 8.0). The protein solution was heated for 3 min at 100 °C and centrifuged at 1500 g for 10 min. Gels were stained with Coomassie Brilliant Blue R-250 and destained with methanol: acetic acid: water (50:10:40) (v/v/v) acetic acid in water.
2.2. Fabrication and heat treatment of LSP
2.6. Measurement of circular dichroism (CD) spectrum
Fabrication of LSP: The LSP was extracted as previously described by Pan et al. (2016). Briefly, the lotus seed powder was mixed with 100 mL of 0.10 mol/L NaCl and constant magnetic stirring (100 rpm) was applied for 1 h at 40 °C. The mixture was then centrifuged for 20 min at 2800 g using a refrigerated centrifuge (Avanti J-26 XP centrifuge, Beckman Coulter, Fullerton, CA, USA). Afterwards, the supernatant was adjusted to pH 4.2 by adding 0.1 mol/L HCl solution (acid precipitation), and then re-centrifuged (4800 g for 25 min). The precipitate obtained was taken to be the crude LSP product. The crude product was washed with deionized water (pH 4.2) four times and subsequently freeze-dried to give the LSP powder. The LSP was mainly composed of globulin, and its protein content was determined to be greater than 90% by Kjeldahl method. Heat treatment of LSP: LSP freeze-dried powder was dissolved in deionized water at 1% (w/v) total solids and the solution was slowly stirred for 4 h at 25 °C and stored overnight at 4 °C to ensure complete hydration. In addition, sodium azide (0.02% w/v) was added to the LSP solution to inhibit bacteria growth. Samples were subjected to different heat treatments (0, 60, 80, 100 °C) for 30 min at pH 7.0 without salt ion. After heat treatment, all samples were quickly cooled in an ice-water bath and then stored in a 4 °C environment until analyzed.
The LSP solution (1% w/v), both before and after heat treatment, was diluted in 10 mmol/L phosphate buffer, pH 7.0 to obtain the 0.02% (w/v) test sample. The CD spectrum was performed using a spectropolarimeter (Jasco 810, Jasco Corp., Tokyo, Japan) in the range 190–240 nm at 25 °C. For this test, a quartz cuvette was used with an optical path of 0.1 cm. The spectral resolution was 0.5 nm, the scan speed was 100 nm/min and the response time was 0.25 s with a bandwidth of 1 nm. Three scans were accumulated and averaged.
2.1. Materials
2.7. Measurement of fluorescence spectrum The test protein solution 0.02% (w/v) was prepared as described in the CD experiment. The intrinsic fluorescence spectroscopy of LSP was performed using a Luminescence Spectrometer (F-4600, Japan) at 25 °C. A quartz cuvette with an optical path of 1 cm was used. The excitation wavelength was 290 nm and the emission spectra ranged from 300 to 460 nm. The excitation and emission slit widths were 2.5 nm and a scan speed of 1200 nm/min was used. 2.8. Dynamic interface properties measurement The interfacial surface pressure (π) and adsorption kinetics of LSP both before and after heat treatment at the oil/water interface were performed using an automated drop tensiometer (Tracker-H, Teclis, France) at 25 °C (Xiong et al., 2018). The LSP solution (1%, w/v) and oil phase (medium chain fatty acid, MCT) were placed in the cuvette and syringe, respectively. The test used the hanging drop method. Before testing, dispersions and oil were allowed to stand for at least 1 h to reach 25 °C. The temperature of the system was maintained constant by circulating water from a thermostat. The oil droplet volume during the test was 10 μL. The oil/water π (mN/m) was calculated with equation (1):
2.3. Measurement of surface hydrophobicity (H0) The surface hydrophobicity of LSP was determined by a fluorescence spectrum assay using 8-anilino-1-naphthalenesulfonic acid (ANSA) as a fluorescent probe (Delahaije, Wierenga, Nieuwenhuijzen, Giuseppin, & Gruppen, 2013). The LSP and ANSA were dissolved in phosphate buffered saline (PBS, pH 7.0, 10 mmol/L) to obtain a 1 mg/ mL and 2.4 mmol/L solution, respectively. Subsequently, aliquots of 50 μL ANSA solutions were added into 5 mL LSP solution for analysis. The excite wavelength was set at 385 nm and the emission wavelength range was 400–650 nm using a fluorescence spectrophotometer (Shimadzu RF-5310PC, Japan). The emission and excitation slits were set at 5 nm and the measurements were performed at 25 °C. The area of the fluorescence spectrum was corrected with the area of the buffer and the relative exposed hydrophobicity was expressed as the area of the sample.
π (mN/m) = γ0-γ
(1)
Where γ0 (mN/m) and γ (mN/m) is the interfacial tension of pure oildistilled water (26 mN/m) and LSP solutions, respectively. The surface tension was determined by drop shape analysis (Benjamins, Cagna, & Lucassen-Reynders, 1996). All measurements were performed for up to 3 h. Generally, there is a rate-determining step, showing relatively low interfacial surface pressures during the first step (Camino, Pérez, Sanchez, Patino, & Pilosof, 2009). The change in interfacial π with adsorption time (t) can be correlated by a modified form of the Ward and Tordai equation:
2.4. Measurement of particle size and zeta potential The zeta potentials of fresh, undiluted samples were determined using a microelectrophoresis instrument (ZS Zetasizer Nano, Malvern Instrument Ltd., UK). The average particle size and polydispersity index 2
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π = 2C0KBT(Dt/3.14)1/2
2012). Additionally, Fig. 1b shows the far-UV CD spectra of untreated and heated LSP. From the intensity of the CD spectra, some changes can be observed in the secondary structure of LSP. The spectra were analyzed using Yang's equation and the proportions of α-helix, β-sheet, β-turn and random coil was ~0%, 82%, 0% and 18%, respectively. These values conflict with those previously reported for native LSP (Zeng, Cai, Cai, Wang, & Li, 2011). This phenomenon could be attributed to the different LSP composition and characterization means used by Zeng et al. (2011) to investigate the secondary structure of LSP. However, the differences in these values between samples was < 1%. It was deduced from these spectra that the secondary structure was not significantly affected by the heat treatment. The effects of heating on the tertiary structure of LSP were examined by the intrinsic fluorescence, which can be reflected by the micro-environmental changes around tryptophan residues of LSP. A maximum fluorescence intensity was observed at 348 nm for both native and heated LSP (Fig. 1c). The fluorescence intensity decreased with an increase in heat treatment temperature from 0 to 100 °C. This variation in fluorescence intensity may be ascribed to the difference in energy transfer efficiency between tryptophan and tyrosine, as well as the chromophores of LSP becoming exposed to solvents due to heat treatment (Xiong et al., 2016). Generally, tyrosine, tryptophan and phenylalanine residues in proteins, particularly tryptophan residues, will be fluorescently polarized in a manner that is highly dependent on protein folding and serve as a tertiary structure for sensitivity monitors (Xiong et al., 2016). Therefore, this result shows that the tertiary structure of LSP had been changed and partly unfolded due to heat treatment, especially for the 100 °C (30 min) heating.
(2)
Where C0 is the concentration in the continuous phase, KB is the Boltzmann constant, T is the absolute temperature and D is the diffusion coefficient. If the adsorption process is controlled by the protein diffusion, a plot of π versus t1/2 will then be linear and the slope of this plot will be the diffusion rate (Kdiff). To obtain interface dilatational parameters, the dynamic interface viscoelasticity of LSP solutions at the oil-water interface were also investigated. The sinusoidal interfacial compression and expansion were performed by changing the drop volume at 10% of the deformation amplitude within the linear regime, the oscillation frequency was 0.1 Hz, the droplet volume was 10 μL. The experiment was started after an equilibration period of 60 s. The number of active and blank was 5 cycles during the experiments. Details of this experiment were described by Perez, Carrara, Sánchez, Santiago, and Patino (2009). The interface dilatational modulus (E) derived from the change in interfacial tension (γ), resulting from a small change in surface area (A), can be described by equation (3): E = dγ/(dA/A) = − dπ/dln (A) = Ed + iEv
(3)
The E is a complex quantity and is composed of real and imaginary parts. The real part of E or storage component is interface dilatational elasticity (Ed). The imaginary part of the E or the loss component is the interface dilatational viscosity (Ev) (Beverung, Radke, & Blanch, 1999). 2.9. Statistical analysis All measurements were carried out in triplicate unless otherwise stated. One-way analysis of variance (ANOVA) with a 95% confidence interval was used to assess the significance of the results obtained. Statistical analysis was performed using SPSS software version 19.0.
3.2. Effects of heat treatment on the size, zeta potential and surface hydrophobicity of LSP
3. Results and discussion
The influence of heating on the average diameter distribution of LSP was conducted by dynamic light scattering (DLS) and the results obtained are presented in Fig. 2a. As expected, all heat treatments resulted in a significant increase in protein mean diameter compared to native LSP, which was due to partial unfolding and aggregation of the protein molecules during heating. Meanwhile, the size of LSP aggregates tended to increase as the heat treatment temperature increased. It is noteworthy that the dimensional distribution curve of the LSP after heating at 80 °C for 30 min showed two peaks, which should be related to the degree of aggregation of the protein under different heating conditions (Liu & Tang, 2014; Wang et al., 2012). This was confirmed by the change of zeta potential and surface hydrophobicity of LSP after heat
3.1. Effects of heat treatment on the structure of LSP The impacts of heat treatment on the primary structure of LSP were evaluated by SDS-PAGE. A comparison of the electrophoretic patterns of the proteins revealed no significant difference between the treated and untreated LSP in terms of their molecular weights, indicating that no significant changes occurred in the primary structure of the protein (Fig. 1a). This suggests that heat treatment does not affect the individual subunits of LSP. A similar phenomenon was also observed in the soybean protein isolate system after heat treatment (Wang et al.,
Fig. 1. Structural characterization of LSP fractions. (a) SDS-PAGE electrophoretic profile of untreated (A) and heated (B–D) LSP. M is the marker. (b) Far-UV CD spectra of untreated (A) and heated (B–D) LSP. (c) Emission fluorescence spectra of untreated (A) and heated (B–D) LSP. The black, red, blue and green lines in Figure b and c are unheated, heat treated at 60 °C, 80 °C, and 100 °C for 30 min, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 3
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Fig. 2. Effect of heating on the particle size (a), zeta potential (b) and surface hydrophobicity (c) of untreated (A) and heated (B–E) LSP. The black, red, blue and green lines in Figure a are unheated, heat treated at 60 °C, 80 °C, and 100 °C for 30 min, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
(Fig. 3A). In addition, the rates of penetration and rearrangement of adsorbed protein molecules at the oil-water interface can be determined by the first-order equation:
treatment (Fig. 2b, c). Compared with natural LSP, the net surface potential of LSP significantly increased after heating (P < 0.05), which is related to the charged amino acid residues on the surface of the protein, except in samples heated at 100 °C for 30 min (P > 0.05). This change is likely related to the charged amino acid residues on the surface of the protein (Delahaije, Gruppen, Giuseppin, & Wierenga, 2014). In addition, the surface hydrophobicity of the LSP was also significantly enhanced after heat treatment (P < 0.05), meaning that hydrophobic residues within the protein molecule became exposed. These results further demonstrate that the degree of unfolding and aggregation of LSP structures are various under different temperature treatments.
ln(πf-πt)/ (πf-π0) = -kit
(4)
Where πf, π0 and πt are the interfacial pressures at the final adsorption time, at the initial time and at any time of each stage, respectively and ki is the first-order rate constant. The application of Eq. (4) to the adsorption of the LSP both before and after heating at the interface is presented in Fig. 3B. Clearly, there were two linear regions in these plots. Generally, the first slope is usually regarded as the rate constant of penetration (KP) and the second slope as the rate constant of molecular reorganization (Kr) (Perez et al., 2009). The rate constants of LSP molecular diffusion (Kdiff), penetration (Kp), reorganization and the equilibrium interfacial pressure (π7200) at the oil-in-water interface are summarized in Table 1. The Kdiff, Kp and π7200 of the LSP without heating was lower than the samples after heat treatment, which might be mainly due to the enhancement of surface hydrophobicity. In general, the interface properties of protein were greatly related to the surface charge, surface hydrophobicity, molecular flexibility and particle size (Morales, Martínez, Ruiz-Henestrosa, & Pilosof, 2015; Wierenga, Meinders, Egmond, Voragen, & de Jongh, 2003; Wierenga, Meinders, Egmond, Voragen, & de Jongh, 2005). The
3.3. Interfacial adsorption and dilatational rheological properties 3.3.1. Adsorption kinetics and structural rearrangements at the oil-in-water interface The interface adsorption behavior of protein molecules at the oil–water interface is mainly influenced by diffusion, conformational rearrange, and actual adsorption (penetration and unfolding) (Dickinson, 2011). Accordingly, the rate at which protein molecules diffuse from the bulk phase to the oil–water interface (Kdiff) can be determined by the linear slope of the interface pressure versus adsorption time curve
Fig. 3. (A) Time dependence of surface pressure (π) for LSP adsorbed layer at the oil-water interface. Kdiff represent diffusion rate. (B) Typical profile of the molecular penetration and rearrangement steps at the oil-water interface for LSP. Kp and Kr represent first-order rate constants of penetration and rearrangement, respectively. The black, red, blue and green dots in the figure are unheated, heat treated at 60 °C, 80 °C, and 100 °C for 30 min, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 4
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Table 1 The diffusion rate (Kdiff), constants of penetration and structural rearrangement at the interface (KP and KR), and surface pressure at the end of adsorption (7200 s, π7200) for LSP pH 7.0. Kdiff (mN/m/s1/2) (LR) A (Unheated) B (60 °C,30min) C (80 °C,30min) D (100 °C,30min)
0.19 0.23 0.25 0.26
± ± ± ±
0.03 0.01 0.02 0.02
(0.9533)b (0.9790)ab (0.9746)a (0.9829)a
Kp × 10−4 (LR) −3.12 −4.02 −4.05 −4.13
± ± ± ±
0.03 0.03 0.02 0.02
Kr × 10−3 (LR) (0.9870)c (0.9976)b (0.9741)b (0.9855)a
−3.33 −3.71 −3.30 −3.18
± ± ± ±
0.03 0.05 0.05 0.03
Π7200 (mN/m) (0.8970) b (0.8853) a (0.9415)b (0.9152)c
17.71 19.60 20.05 20.13
± ± ± ±
0.06c 0.10ab 0.06a 0.06a
LR is an abbreviation for linear regression coefficients. Different letters in the same column indicate significant differences (P < 0.05).
interfacial adsorption layer, thereby exhibiting a lower viscoelastic property (E value). On the other hand, as more LSP molecules adsorb to the interfacial layer, they may cause the already adsorbed protein molecules to dissociate from the interface and contribute to the decreasing trend in the E value. Furthermore, the dynamic dilatational elastic moduli (Ed) of interfacial layers of all samples are presented in Fig. 4B. Clearly, the Ed values gradually increased due to the adsorption of protein at the interface and almost showed the same trend as E. After adsorption for 120 min, the Ed values were close to the surface dilatational modulus (E values) and the dilatational viscosity values were low (data not shown). This result suggested that the interface absorbed layer mainly exhibited the elastic behavior.
increase of protein particle size and surface net potential could play a negative role for the diffusion and adsorption rate of protein in oilwater and air-water interfaces. In all cases, the interfacial surface pressure increased with adsorption time (Fig. 3A), which could be ascribed to the LSP adsorption at the interface (Perez et al., 2009; Wan, Wang, Wang, Yuan, & Yang, 2014). On the other hand, Kr for the adsorbed LSP was considerably higher than Kp, suggesting the importance of the structural rearrangement of adsorbed LSP at the interface to film formation rather than their penetration. 3.3.2. Dilatational rheological properties at the oil-in-water interface The stability of the protein emulsion can be predicted by studyingthe viscoelastic properties of the protein-adsorbed layer at the oil–water interface (Murray, 2002). Therefore, the surface E as a function of interfacial π in the surface layer for the adsorption of LSP before and after heating is presented in Fig. 4A. The E-π curve and its slope reflect the equilibrium state of the adsorbed protein at the interface. If the increase of E is only related to the adsorption amount of the surfactant on the interface, the E data of all the systems should be approximately the same in the E-π curve. It can be observed from Fig. 4A that E increased with the increase of the π value for all samples, indicating that there was interaction between the adsorbed protein molecular residues which were gradually increasing. The slopes of the E-π curves were greater than 1, which demonstrates a non-ideal adsorption state, suggesting that there was a strong interaction between the adsorbed protein molecules. Similar results were also observed in other heated protein systems (Liu & Tang, 2014; Wang et al., 2012). However, when the π was greater than 18 mN/m, the E value exhibited a decreasing trend and the heat-treated samples had lower E values than the native LSP. This result can be explained as the LSP size and the surface electrostatically increased due to the heat treatment, resulting in a decrease in the interaction between the protein molecules of the
4. Conclusion Heat treatment induced partial de-folding and aggregation of the LSP structure, exhibiting greater average diameter and surface hydrophobicity. However, heating showed multiple effects on the surface potential of LSP. Moderate heat treatment (60 and 80 °C) can significantly increase the surface net potential of LSP, while heating at 100 °C for 30 min has no significant effect. In addition, the diffusion and permeation rate of LSP at the oil-water interface were increased after heat treatment and exhibited higher surface pressure values. However, the viscoelastic properties of the interface adsorption layer have not been enhanced and the interfacial rheological behavior dominated by the elastic modulus has been demonstrated. It is speculated that the above findings may be related to the change of LSP structure caused by heat treatment.
Fig. 4. (A) Surface dilatational modulus (E) as a function of surface pressure (π) for LSPs at the oil−water interface. (B) Time-dependent dilatational elasticity (Ed) for LSPs adsorbed layers at the oil-water interface. The black, red, blue and green dots in the figure are unheated, heat treated at 60 °C, 80 °C, and 100 °C for 30 min, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 5
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Declaration of interest statement
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