Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Lipo-Dipeptide as an Emulsifier: Performance and Possible Mechanism Wenhui Lv,†,‡ Tan Hu,†,‡ Ahmed Taha,†,§ Zhongkun Wang,†,‡ Xiaoyun Xu,†,‡ Siyi Pan,†,‡ and Hao Hu*,†,‡ †
College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, People’s Republic of China Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Ministry of Education, People’s Republic of China § Department of Food Science, Faculty of Agriculture (Saba Basha), Alexandria University, Alexandria 21531, Egypt ‡
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S Supporting Information *
ABSTRACT: A lipo-dipeptide (C13-lysine-arginine, C13-KR) was designed as a potential emulsifier with good emulsifying properties under acidic condition. Compared with two traditional emulsifiers (whey protein isolate and Tween 80), C13-KR emulsion had the minimum mean size but the highest zeta potential (around +100 mV). Moreover, C13-KR emulsion showed better stability against environmental stresses, such as high salt concentrations and high temperature. The C13-KR particles had the fastest move rate around 400 Hz when it attained an equilibrium state. Furthermore, C13-KR emulsifier could sharply reduce the interfacial tension and had the lowest tension value at the oil/water interface. The interfacial tension of C13-KR emulsifier was only 3.6 mN/m (0.5% w/v). In conclusion, the lipo-dipeptide C13-KR could be considered as an emulsifier to produce emulsion under acidic condition. KEYWORDS: lipo-dipeptide C13-KR, emulsifiers, emulsion stability, environmental stresses, ultrasound
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INTRODUCTION Emulsifier is a key component for stable emulsion production, which is widely used in food, cosmetic, encapsulation, and biomedicine industries.1−4 However, traditional emulsifiers may have some disadvantages, such as instability under environmental stresses (acidic environment, high temperature, and high salt concentrations),5−8 which limit their further applications. Hence, it is required to develop effective emulsifiers with good stability to match with various applications. Peptides as emulsifiers have attracted much attention due to their superior functionalities and chemical diversities,9−12 which could be used to design emulsifiers with different applications. For example, Wibowo and co-workers13,14 reported that AM1 (Ac-MKQLADS LHQLARQ VSRLEHACONH2) was designed to stabilize emulsion with high stability. Mondal et al.15 demonstrated that SHR-FLLF (H2N-Phe-Aib-Leu-Ala-Aib-Leu-Phe-OH) with a rigid backbone could afford long-term emulsion stability, forming highly stable emulsions compared with traditional emulsifiers such as Tween 20 and SDS. Recently, it was found that natural short peptides as emulsifiers could also stabilize emulsions. Scott et al.16 reported that three tripeptides lysine-tyrosine-phenylalanine (KYF), lysine-phenylalanine-phenylalanine (KFF), and lysine-tyrosine-tryptophan (KYW) could stabilize emulsions due to the fact that tripeptides formed nanofibers networks at oil droplets surfaces. However, it was observed that the stability of those natural short peptides emulsions was significantly influenced by the surrounding environment, such as temperature. Therefore, some chemical groups, such as fluorenylmethoxycarbonyl (Fmoc) and naphthalene (Nap), © XXXX American Chemical Society
were used to modify natural short peptides. These modifications could improve the stability of short peptides emulsions under different environmental stresses. Ulign and co-workers17,18 showed that Fmoc-modified dipeptide (FmocYL) emulsion exhibited good stability under different environment conditions such as high temperature and high salt concentration. Furthermore, Fernando Aviño et al.19 also reported that naphthalene-protected diphenylalanine (2NapFF) hydrogel could self-assemble at the oil/water interface to form long-term stable emulsion. However, the potential toxicity of these chemical groups (Fmoc, Nap) may limit their further applications. Compared with the aforementioned modified peptides, carbon-chain-modified shortpeptide emulsifier could be considered as a low-toxicity emulsifier. Emulsions are widely applied in the food and beverages industries. Moreover, emulsions are used as delivery systems that can encapsulate various lipophilic active components. However, many food systems are in acidic condition,20,21 which reduces the stability of emulsions formed by some traditional emulsifiers. For example, food protein-based emulsifiers possess superior emulsification properties, but many of them are sensitive to acidic environment, which is close to their isoelectric points. Hence, developing emulsifiers that can stabilize emulsions in acidic environment is vital to the food industry. Received: March 18, 2019 Revised: May 7, 2019 Accepted: May 21, 2019
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DOI: 10.1021/acs.jafc.9b01721 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Particle Size and Zeta Potential Analysis. The MasterSizer 2000 equipment (Malvern Instruments, Worcestershire, UK) was used to measure the particle sizes of freshly produced emulsions.26 The dispersion medium refractive index and the MCT oil refractive index were 1.330 and 1.440, respectively. Besides, the volume mean diameter (d4,3) was used to measure the particle sizes, which was sensitive to larger droplets, especially when emulsions were formed by poor emulsifiers.27 The zeta potential values of emulsions (diluted 50 times) were measured by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments) according to the method of You et al.28 Creaming Index. To test emulsion stability, emulsion samples formed by different emulsifiers with concentrations of 0.25% and 0.5% (w/v) were characterized in acidic environment. Freshly prepared emulsions were transferred into 1 mL glass tubes and stored at room temperature for 2 weeks. During storage, emulsions may separate into the cream layer at the top. The height (mm) of the cream layer (Hc) and the total height (mm) of the emulsion (Ht) were measured by a ruler. Then the creaming index (CI) was calculated after 1, 7, 15 days H according to the equation CI % = Hc × 100.29
In this study, a lipo-dipeptide emulsifier C13-lysine-arginine (C13-KR) was designed (Figure S1). Compared with natural short peptides (tripeptides), C13-KR emulsions may have better stability. Moreover, compared with long peptides (21 or 7 amino acids) emulsifiers, the primary and advanced structures of C13-KR were simple (composed only two amino acids and a carbon chain). Therefore, it was easier to synthesize and analyze C13 -KR, which could provide advantages for potential industrial production. Furthermore, compared with peptides modified by some chemical groups (fluorenylmethoxycarbonyl and naphthalene), C13-KR was composed of 13 carbon atoms at the N-terminus as the hydrophobic group. The carbon chain might attain a stronger anchor to the oil phase to promote adsorption at the oil/water interface.22 Therefore, the peptide C13-KR may achieve better surface activity to enhance the stability of emulsions. To the best of our knowledge, little is known about the emulsifying properties and mechanisms of lipo-dipeptides formed emulsions. In this study, the physicochemical properties of ultrasound-induced emulsions stabilized by C13-KR, WPI, and Tween 80 were investigated. This study could provide some basic understanding of using lipo-dipeptide as a potential emulsifier.
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Emulsion Stability under Environmental Stresses. Ionic Strength. Emulsions were prepared using different emulsifiers at concentration of 0.5% (w/v). To investigate the effects of salt concentrations on emulsion stability, fresh emulsions were mixed with different concentrations of NaCl solutions (50, 100, and 200 mM).30 The vortex mixer was used to mix emulsion and salt solution (volume ratio = 1:1) for 3 min. Then samples were standing for 30 min. After that the particle size (d4,3) of the mixture was measured.31 Temperature. To investigate the effect of heating treatment on emulsion stability, fresh emulsions (0.5% w/v) were heated at 90 °C for 15 and 30 min.32 After heating, samples were cooled to room temperature; then particle sizes (d4,3) were measured. Emulsion Microstructure. Fluorescence Microscopy. The microstructure of freshly prepared emulsion was observed by fluorescence microscopy (DM3000 microscope, Leica, Germany).33 Nile blue and nile red dyes were mixed (1:1, v/v) and used to stain the aqueous and oil phase, respectively.34,35 Before analysis, 1 mL of fresh emulsion and 10 μL of dyes mixture were mixed. Then 5 μL of emulsion was located on the microscope slide to observe the microstructure of the emulsion sample. Optical Microscopy. Optical microscopy (Nikon Eclipse Ti-S, Nikon Instruments) was used to observe the changes of emulsion morphology. The microstructure of the emulsion was observed by using a 40× magnification lens.36 Microrheological Properties. Fluidity Index (FI) of Emulsifier. The particle Brownian motion was measured by a Rheolaser Master (Formulaction, France).37 The fluidity index (FI) value of the sample was obtained using the software RheoSoft Master (version 1.4.0.0). The emulsifier concentration was 0.25% (w/v). Elasticity Index (EI) of Emulsion. The elasticity index of the emulsion was measured by the Rheolaser Master which was based on diffusing wave spectroscopy (DWS).38 In this experiment, emulsion samples were placed into 4 mL flat-bottomed cylindrical glass tubes. The elasticity index (EI) value of emulsion was tested after storing for 1, 5, and 13 days. The emulsifier concentration was 0.25% (w/v). Interfacial Tension Measurement. To compare the interfacial rheology properties of 3 different emulsifiers, the interfacial tension (γ) of different emulsifiers (C13-KR, WPI, Tween 80) was characterized using a Tracker drop tensiometer (Tracker Teclis/IT Concept, France).39 The test was carried out by the pendant drop method. MCT oil was put in a glass cuvette, and the emulsifiers dispersions were inserted into the oil phase using a syringe. Then the interfacial tension (γ) of the drop was analyzed using the Laplace equation. All of the measurements were performed at 25 °C for 7200 s with an initial drop volume of 15 μL. Statistical Analysis. All tests were repeated in triplicate using freshly prepared samples. Statistical analyses were performed in triplicate by using SPSS 22 software. Average and standard deviations were evaluated using one-way ANOVA followed by Duncan’s test. P value < 0.05 was considered statistically significant.
MATERIALS AND METHODS
Materials. Lipo-dipeptide C13-KR (99.87%) was synthesized by China Peptides Co., Ltd. (Shanghai, China). The physicochemical properties of the lipo-dipeptide C13-KR are shown in Table S2. C13KR was only soluble in acidic condition. Tween 80, citric acid monohydrate, and sodium chloride were purchased from Sinopharm Chemical Reagent Co., Ltd. Whey protein isolate (WPI) and medium-chain triglyceride (MCT) oil were obtained from the Shanghai Yuanye Bio-Technology Co., Ltd. Nile red and nile blue dyes were bought from Sigma Chemical Co., St. Louis, MO, USA. Lipo-Dipeptide Synthesis, Purification, and Analysis. Lipodipeptide (C13-KR) was synthesized by the method of Fmoc solidphase synthesis23−25 (the chemical structure of C13-KR is shown in Supporting Information Figure S1). The peptide was synthesized from C-terminal to N-terminal. 2-Chlorotrityl chloride resin was used to make peptide C-terminal amidated. First, Fmoc-Arg-OH was coupled with resin. After deprotection of the Fmoc group with 20% piperidine in DMF, the Fmoc-Lys-OH was coupled. Lastly, tridecanoic acid was introduced to complete the designed peptide sequence. All coupling reactions were activated by O-benzotriazoleN,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU). After completing the chain assembly, the peptide was cleaved from resin by cleavage reagent. Cleavage was performed with 95% TFA, 2% TIS, 2% EDT, and 1% H2O. Filtration and evaporation were used to remove cleavage mixture. Then the peptide was collected and dried. The results of HPLC purification and ESI-MS analysis are given in the Supporting Information (Figure S2, Table S1, and Figure S3). The purity of C13-KR was 99.87%. Emulsion Preparation. To formulate the emulsion, MCT oil and aqueous phase were mixed at specific conditions. C13-KR, WPI, and Tween 80 were dissolved in 1% (w/v) citric acid solution. The final concentrations of emulsifiers were 0.25−1% (w/v). The final concentration of MCT oil was 5% (w/v). A 1 mL amount of the mixture was added to a centrifuge tube (1.5 mL). Then the mixture was vortexed for 3 min by a vortex mixer (SCI LOGEX). Subsequently, the mixtures were subjected to ultrasound emulsification (with 0.30 cm diameter probe, Ning Bo Scientz Biotechnology Co, Ltd.) at 30% amplitude. The ultrasound power was 285 W, and ultrasound intensity was 0.574 W cm−3 in the current study. The sonication pulse duration was set as on time 2 s and off time 2s. Emulsions were treated for 10, 20, and 40s (effective sonication time with omitting pulse time). B
DOI: 10.1021/acs.jafc.9b01721 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 1. Particle size of emulsions influenced by different sonication times and different emulsifiers (A), different emulsifier concentrations and different emulsifier types (B), and fluorescence micrographs of emulsions stabilized by different emulsifiers at a concentration of 0.5% (w/v) and sonication time of 20 s: (C) C13-KR, (D) Tween 80, and (E) WPI. Scale bar represents 5 μm. Labels “A, B, C” and “a, b, c” in A mean that the size of emulsions prepared by the same sonication time are significantly different with different emulsifiers (p < 0.05) and the size of emulsions prepared by the same emulsifier are significantly different with different emulsifier concentrations, respectively. Labels “A, B, C” and “a, b, c” in B mean that the size of emulsions prepared by the same emulsifier concentration are significantly different with different emulsifiers (p < 0.05) and the size of emulsions prepared by same emulsifier are significantly different with different emulsifier concentrations, respectively.
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fluorescence images of emulsions stabilized by different emulsifiers. It was obvious that emulsions formed by WPI had the largest oil droplet size, but C13-KR had the lowest particle size. These findings were consistent with the droplet size data (Figure 1A and 1B). From the above-mentioned information, it could be observed that C13-KR was the most effective emulsifier among all of the emulsifiers used in this study. Creaming Index of Emulsion. The creaming index (CI) could provide indirect information about emulsion stability.41 If the CI value is lower, the emulsion will be more stable. It was observed that emulsion stabilized by C13-KR had the lowest CI (Figure 2A), showing the highest stability among all emulsions. On the other hand, WPI showed the highest value of CI, which revealed the lowest stability. Figure 2B−D shows images of emulsions stored for 2 weeks. It was obvious that C13-KR emulsion showed the best stability, while WPI emulsion was relatively unstable. The reason could be due to the fact that C13-KR emulsifier had better surface activity and lower interfacial tension than the other two emulsifiers, producing smaller droplet size emulsions. Thus, C13-KR showed better stability of emulsions than the traditional emulsifiers (Tween 80 and WPI). Similar results were reported by Kelley et al.,41 who found that the increase of the droplet sizes could facilitate flocculation of oil droplets in emulsions. The result of creaming stability was consistent with the interfacial tension and the droplet size (Figures 1 and 9). Elasticity Index of Emulsion. In order to further understand emulsion stability in acidic condition, the elasticity index (EI) values of different emulsions (with 0.25% w/v
RESULTS AND DISCUSSION Emulsion Particle Size and Microstructure. In order to study the emulsifying properties of the lipo-dipeptide (C13KR), Tween 80 and WPI were chosen as control. Tween 80 and WPI are traditional emulsifiers, which are widely used in various fields.2,4,40 Figure 1 shows the particle sizes of emulsions formed by different emulsifiers. As shown in Figure 1, lipo-dipeptide C13-KR emulsion had the lowest particle size (d4,3 < 1 μm) among all of the emulsifiers, suggesting that C13KR had the highest emulsification activity. The reason for this result could be explained as follows. Compared to the protein (WPI), C13-KR had a smaller molecular weight, which could quickly move to the interface to attain fast adsorption. Moreover, the hydrophobic carbon chain of lipo-dipeptide C13-KR could provide better anchoring at the oil/water interface than Tween 80 to enhance surface activity. This could be a reason for the low particle size of C13-KR emulsions compared to that of Tween 80 emulsions. As shown in Figure 9, the C13-KR could quickly adsorb at the oil/water interface and had a lower interfacial tension value than the other two emulsifiers. The relatively rapid adsorption of C13-KR could facilitate formation of emulsion with small droplet size.22 Moreover, a longer sonication emulsifying time (Figure 1A) decreased the particle size of all emulsions significantly. Furthermore, the particle sizes of all studied emulsions decreased with increasing emulsifier concentration (Figure 1B), suggesting that a higher emulsifier concentration could provide more emulsifier molecules to absorb onto droplet surfaces to stabilize emulsion. Figure 1C−E shows the C
DOI: 10.1021/acs.jafc.9b01721 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 2. Creaming index (%) of emulsions stabilized by different emulsifiers with concentrations of 0.25% (w/v) and 0.5% (w/v) (A). Visual observation of creaming stability of emulsions stabilized by different emulsifiers (KR, C13-KR; T, Tween 80; W, WPI) after (B) 1 day, (C) 7 days, and (D) 15 days.
WPI showed further decreasing trend after storage for 13 days. However, EI of C13-KR-stabilized emulsion reduced during the first 5 days and then remained relatively unchanged. Most importantly, there were no significant differences among the EI values of emulsions formed by the three emulsifiers after storage for 5 days. On the basis of these results, it could be demonstrated that the EI of C13-KR emulsion had the minimum change with increasing storage time. It could be suggested that the elastic film structure of C13-KR emulsion had a lower damaging rate than those of the other emulsions during storing. This could be a reason for the higher stability of C13-KR emulsions during storage. The result of EI is in agreement with the creaming stability. Zeta Potential. A higher surface charge resulted in stronger electrostatic repulsion around oil droplets to prohibit emulsion flocculation and phase separation.43 Therefore, the zeta potential values of fresh emulsions were measured. As shown in Figure 4, emulsion formed by C13-KR had the highest zeta potential around +100 mV, suggesting that it had the highest electrostatic repulsion. However, the zeta potential of Tween 80 emulsion showed the lowest surface charge value. The reason could be due to the fact that Tween 80 is a nonionic surfactant. A similar result was mentioned by Yang et al.,44 who showed that the surface charge of Tween 80 reached zero at pH around 3.5. Furthermore, Song et al.45 reported that the surface charge of the emulsion droplets could influence the emulsion stability due to its electrostatic repulsion effects.
emulsifier concentration) were measured during storage. Figure 3 shows the microrheology characteristics (EI) of different emulsions. A higher EI value may prohibit droplets sedimentation and phase separation to maintain emulsion stability.42 As presented in Figure 3, emulsion stabilized by WPI had the highest EI value and emulsion formed by C13-KR had the lowest EI value in the first day. Interestingly, the EI of WPI emulsion decreased sharply after storage. Moreover, EI of
Figure 3. Elasticity index (EI) values of emulsions stabilized by different emulsifiers (0.25% w/v) as a function of storage time. Labels “a, b, c” mean that the value of emulsion EI stabilized by the same emulsifier is significantly different with storage time (p < 0.05). Labels “A, B, C” mean that the value of emulsion EI stabilized by the same storage time is significant with different emulsifier (p < 0.05). D
DOI: 10.1021/acs.jafc.9b01721 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 4. Zeta potential values of emulsions affected by different sonication times and different emulsifiers (A), and zeta potential values of emulsions affected by different emulsifier concentrations and different emulsifier types (B). Labels “A, B, C” and “a, b, c” in A means that the zeta potential values of emulsions prepared by the same sonication time are significantly different with different emulsifiers (p < 0.05) and the zeta potential values of emulsions prepared by the same emulsifier are significantly different with different sonication time, respectively. Labels “A, B, C” and “a, b, c” in B mean that the zeta potential values of emulsions prepared by the same emulsifier concentration are significantly different with different emulsifiers (p < 0.05) and the zeta potential values of emulsions prepared by the same emulsifier are significantly different with different emulsifier concentrations, respectively.
Figure 5. Influence of ionic strength (50, 100, and 200 mM NaCl) on the particle size distribution and volume mean diameter (d4,3) of emulsions stabilized by different emulsifiers at a concentration of 0.5% w/v: (A) C13-KR, (B) Tween 80, and (C) WPI.
Figure 6. Influence of heat treatment (90 °C) for 15 and 30 min on the particle size distribution and volume mean diameter (d4,3) of emulsions stabilized by different emulsifiers at a concentration of 0.5% w/v: (A) C13-KR, (B) Tween 80, and (C) WPI.
Moreover, Mondal et al.15 designed a helical peptide with high zeta potential value to form stable emulsion at pH below 2. Therefore, the high surface charge of C13-KR emulsion could be considered as an important factor for improving its stability. This could be one reason why emulsion formed by C13-KR had better creaming stability (Figure 2). Effect of Ionic Strength on Emulsion Stability. The effect of ionic strength on the particle size of the emulsion is shown in Figure 5. Obviously, C13-KR emulsion showed the smallest particle size and the highest stability compared with the other two emulsions. As shown in Figure 5A, the particle
size of C13-KR emulsion had no significant change with an increase of salt concentration. However, particle sizes of Tween 80 and WPI emulsions increased sharply with increasing salt concentration (Figure 5B and 5C). C13-KR had a higher surface charge (Figure 4), which could provide enough electrostatic repulsion to overcome the attractive forces and could weaken the salt-induced electrostatic screening to increase the emulsion stability against ionic strengths.46 Effect of Temperature on Emulsion Stability. Destabilization of emulsion at high temperature limited its application. E
DOI: 10.1021/acs.jafc.9b01721 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 7. Influence of heat treatment (90 °C for 30 min) on the microstructure change of emulsions stabilized by different emulsifiers (0.5% w/v) compared to the nonheated samples.
temperature could lead to the conformational changes of the structure of WPI.49,50 However, the structure of C13-KR was relatively simple, which contained alkyl chain in contact with oil phase and hydrophilic amino acids in contact with aqueous phase at the oil/water interface. Thus, high temperature may not be able to influence its conformational structure significantly, which could improve the heat stability of C13KR-stabilized emulsions. Moreover, the change of emulsion microstructure after heating was observed by optical microscopy. Figure 7 shows the effect of heat treatment (90 °C, 30 min) on the morphology of emulsions. As shown in Figure 7, heat treatment did not change the microstructure of C13-KR emulsion obviously. However, the mean size of the emulsion formed by the other two types of emulsifiers increased markedly after heating at 90 °C for 30 min. These results were consistent with the particle size data (Figure 6). Fluidity Index of Emulsifier. The motion rate of different emulsifier particles was measured by the method of microrheology. In this part, the fluidity index (FI) values of the three emulsifiers (C13-KR, WPI, and Tween 80) particles were tested. The FI represents the mobility of the emulsifier particles. From Figure 8 it could be found that C13-KR emulsifier had the highest FI value (around 400 Hz) when it
Thus, it was important to investigate the stability of emulsions at high-temperature conditions. As shown in Figure 6A, the particle size of C13-KR emulsion did not remarkably change after heating at 90 °C for 30 min, suggesting that C13-KR emulsion had favorable thermal stability. However, emulsions stabilized by Tween 80 and WPI were sensitive to heat treatment as the results show in Figure 6B and 6C. It was concluded that C13-KR emulsions exhibited desirable stability at thermal treatment in contrast to emulsions stabilized by traditional emulsifiers (Tween 80 and WPI). One reason for the good thermal stability of C13-KR emulsion could be due to the fact that C13-KR emulsion had a high zeta potential value, leading to forceful electrostatic repulsion to prevent aggregation of oil droplets when heating samples. Another reason was probably due to the difference of emulsifier conformational structure changes at the oil/water interface under elevated temperature. A similar result was observed from Zhu et al.,7 who demonstrated that heat treatment of Tween 80 emulsion could lead to instability of the emulsion. That may be due to dehydration of the head groups of nonionic surfactants (i.e., Tween 80) and alteration of optimum curvature at the oil/water interface.47,48 Similarly, heating of WPI could influence the emulsion stability. That may be due to the fact that heating beyond its thermal denaturation F
DOI: 10.1021/acs.jafc.9b01721 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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could be diverse. First, from Figure 8, C13-KR particles had the highest motion rate, suggesting that C13-KR emulsifier moved faster to the interface. Moreover, compared with WPI and Tween 80, the advanced structure of C13-KR was relatively simple; therefore, C13-KR did not need too much time to change its conformational structure at the oil/water interface. Thus, C13-KR could achieve ultralow interfacial tension at the oil/water interface instantaneously. This may be considered as an important factor for the good emulsifying property of C13KR (Figure 1). Moreover, in order to explore the influence of emulsifier concentration on the interfacial tension, different concentrations of C13-KR (0.25 to 1% w/v) were selected. As shown in Figure 9B, the interfacial tension decreased gradually when the concentration of C13-KR was 0.25% (w/v). The reason could be due to the fact that the emulsifier interface adsorption decreased the interfacial tension. Furthermore, when the concentration of C13-KR was increased to 1%, the adsorption rate at the oil/water interface was increased but the interfacial tension was decreased to around 3.4 mN/m. The proposed mechanism of C13-KR for stabilizing emulsion is shown in Figure 10. Compared with Tween 80 and WPI, C13-KR particles had higher mobility, which could promote movement from the aqueous phase to the oil/water interface and could increase the adsorption rate. Furthermore, C13-KR emulsifier could instantaneously reduce the oil/water interfacial tension to the lowest value, which could be considered as another important reason for its good emulsifying property. As for emulsion stability, emulsion formed by C13-KR showed better stability than those of WPI and Tween 80. One reason could be due to the fact that emulsion formed by C13-KR had the smallest particle size (d4,3 < 1 μm). Moreover, C13-KR emulsion had the highest zeta potential around +100 mV, which provided strong electrostatic repulsion that could inhibit the creaming and aggregation of oil droplets. Furthermore, the conformational structure of C13-KR was relatively simple, which might be not sensitive to high temperature. Therefore, C13-KR emulsion had better stability during storing and remained stable against environmental stresses.
Figure 8. Fluidity index (FI) of different emulsifiers with a concentration of 0.25% w/v.
attained equilibrium state, meaning that the particles of C13-KR had the highest movement rate. Moreover, the FI value of Tween 80 was the lowest, which represents the lowest movement rate. The reason for the high motion rate of C13KR could be due to the fact that C13-KR had the highest zeta potential value to increase intermolecular electrostatic repulsion.51 The higher FI value of C13-KR particles reflected its higher mobility than other emulsifier particles. During the emulsifying process, C13-KR could move faster from the aqueous phase to the o/w interface and quickly reduce interfacial tension. These findings were consistent with the results of dynamic interfacial tension (Figure 9). Dynamic Interfacial Tension. Formation of emulsions requires emulsifiers to adsorb rapidly at the oil droplet surfaces to reduce the interfacial tension.52 Therefore, the interfacial tension values of different emulsifiers at the oil/water interface were measured to further understand the emulsifying mechanism. As shown in Figure 9A, WPI and Tween 80 (traditional emulsifiers) gradually decreased the interfacial tension from around 10.0 to 8.5 mN/m and from around 6.7 to 4.0 mN/m, respectively. In contrast, C13-KR decreased the interfacial tension at the oil/water interface instantly. From Figure 9A, the interfacial tension value between C13-KR solution (0.5% w/v) and MCT was around 3.6 mN/m and remained relatively unchanged over the period of test time. It could be observed that C13-KR emulsifier could instantaneously obtain the equilibrium tension value and attained the lowest interfacial tension. Therefore, the adsorption kinetics of C13-KR at the oil/water interface was different from the other two emulsifiers, suggesting that the adsorption mechanisms
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CONCLUSIONS A lipo-dipeptide, C13-KR, was designed as an emulsifier to stabilize emulsions. Compared with Tween 80 and WPI, C13KR showed better emulsification properties and formed more stable emulsions. C13-KR particles could move rapidly to the
Figure 9. Dynamic oil/water interfacial tension of different emulsifiers (C13-KR, Tween 80, and WPI) with a concentration of 0.5%w/v (A). Influence of different C13-KR concentrations (0.25%, 0.5%, and 1% w/v) on the oil/water interfacial tension (B). G
DOI: 10.1021/acs.jafc.9b01721 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 10. Proposed schematic diagram of the possible emulsification mechanism of lipo-dipeptide C13-KR.
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oil/water interface and reduce interfacial tension. Moreover, lipo-dipeptide C13-KR had the lowest interfacial tension among all emulsifiers. Furthermore, compared with traditional emulsifier (WPI and Tween 80), C13-KR emulsions had better stability against environmental stresses, such as varied salt concentrations and high temperature. In conclusion, this study demonstrated that the lipo-dipeptide showed good emulsifying property and could be considered as a potential emulsifier. However, the proposed emulsifying mechanism could be different from those of WPI and Tween 80. Further works should be carried out to study the safety of the lipo-dipeptide.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b01721. Chemical structure of lipo-dipeptide C13-KR; HPLC analysis of lipo-dipeptide C13-KR; HPLC analysis report of lipo-dipeptide C13-KR; some physicochemical properties of C13-KR; MS analysis of lipo-dipeptide C13-KR (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Ahmed Taha: 0000-0002-2239-291X Hao Hu: 0000-0003-4653-4959 Funding
The authors acknowledge project 31871726 supported by the National Nature Science Foundation of China (NSFC) and Project 2662017JC015 supported by the Fundamental Research Funds for the Central Universities, China. Notes
The authors declare no competing financial interest. H
DOI: 10.1021/acs.jafc.9b01721 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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