Maillard-Reaction-Functionalized Egg Ovalbumin Stabilizes Oil

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Maillard-Reaction-Functionalized Egg Ovalbumin Stabilizes Oil Nanoemulsions Gang Liu,* Dan Yuan, Qi Wang, Wanrong Li, Jie Cai, Shuyi Li, Olusola Lamikanra, and Xinguang Qin* College of Food Science and Engineering, Wuhan Polytechnic University, Wuhan, Hubei 430023, People’s Republic of China ABSTRACT: Egg white proteins are an excellent source of nutrition, with high biological and technological values. However, their limited functional properties prevent their widespread industrial applications. In this study, the ovalbumin functionality was improved via glycation by Maillard reaction with D-lactose. The free amino groups and sodium dodecyl sulfate−polyacrylamide gel electrophoresis profile were determined, confirming that glycation occurred between ovalbumin and lactose. The emulsification of the conjugate was 2.69-fold higher than that of ovalbumin at pH 7.0 after glycation. The thermal stability also improved remarkably. The glycated protein products were used to form an oil−water nanoemulsion for polymethoxyflavone-rich aged orange peel oil. The resulting nanoemulsion showed good pH, thermal, and storage stabilities. KEYWORDS: ovalbumin, Maillard reaction, functional property, nanoemulsion, stability

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polymethoxyflavones (PMFs), which are flavones containing numerous methoxy groups on a 15 carbon benzo-γ-pyrone skeleton structure with a C4-position carbonyl group.14 PMFs exhibit biological functionalities, such as anti-inflammation and protection against cardiovascular diseases. Nevertheless, the bioavailability and efficacy of PMFs are attenuated by their poor aqueous solubility as a result of their lipophilic chemical structure.15 Nanoemulsion technology is an excellent method to encapsulate oil droplets and, consequently, increase their solubility and stability in the aqueous phase.16 In the present study, we used the ovalbumin glycation product (OGP) as a surfactant to form AOPO-in-water nanoemulsions and optimized the formula for nanoemulsion preparation. Proteinstabilized oil-in-water (O/W) emulsions have been developed and used to deliver hydrophobic natural products in functional food applications. In this case, these emulsions are likely vulnerable to various chemical and physical factors, such as variations in the temperature, pH, ionic strength, and long-term storage.17 The physical and chemical stabilities of emulsion particles under severe conditions can remarkably affect how the delivered bioactive compounds are digested and adsorbed.18,19 As such, proteins, such as whey protein and soy protein isolates, in emulsions may undergo unstable reactions in the gastric region.20 Polysaccharides conjugated to proteins can improve their stability against thermal treatment.21 Covalently bonded conjugates are highly stable in adverse conditions and tolerant to changes in the pH, temperature, and ionic strength.22 This study focused on (1) the Maillard reaction between ovalbumin and D-lactose carried out in a controlled dry state, (2) the characterization of the emulsification, stability, and thermal and absorption properties on the oil−water interface of the resultant conjugates after Maillard reaction, and (3) the use

INTRODUCTION Egg white protein (EWP), an aqueous-rich medium, is mainly composed of ovalbumin,1 ovotransferrin, and ovomucoid, with isoelectric points of 4.5−4.8, 5.8−6.0, and 3.9−4.3,2 respectively. In addition, lysozyme, G2 and G3 globulins, and ovomucin are the other major protein compounds in egg white. 3 Ovalbumin is a 45 kDa phosphoglycoprotein considered as the most abundant protein in EWP and accounts for approximately 54% of the total protein content. 4 Ovalbumin, as a single peptide chain, consists of 385 amino acids, and 50% of them are hydrophobic.5 In numerous studies, the functional properties of ovalbumin have been improved using approaches involving chemicals and enzymes.4 However, only a few methods are suitable to the food industry as a result of safety issues.6,7 Maillard reaction is an environmentally friendly and safe approach performed under mild conditions with well-regulated temperature and relative humidity (RH).8 This reaction enhances the performance of several proteins, including whey protein isolate, soy protein isolate, EWP, and β-lactoglobulin. Ovalbumin has been studied to enhance its properties by glycation under a dry condition of 50−80 °C with glucose, aldohexose, and ketohexose9 and at an aqueous condition of 20−100 °C.10 The wet approach involves heating of protein and saccharide aqueous solution for several days, which results in microbiological spoilage. The dry heating approach, with a higher reaction efficiency than that of the wet approach, has been investigated extensively.11,12 Some rheology, solubility, foaming, and heat stability improvements were reported under a number of acidic conditions. However, in view of the protein drink application, further work is needed to prepare heat-stable and transparent protein dispersions under varied pH values and absorption properties on the oil−water interface. Glycation reduces an essential amino acid.13 Glycation modification can potentially improve the functionality of ovalbumin. Therefore, the variations in functional property parameters of glycated ovalbumin should be studied. Aged orange peel oil (AOPO) is a typical essential oil used in food, cosmetic, and drug industries. AOPO is rich in © XXXX American Chemical Society

Received: Revised: Accepted: Published: A

January 25, 2018 March 29, 2018 April 5, 2018 April 5, 2018 DOI: 10.1021/acs.jafc.8b00423 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

mixed with 2 mL of soybean oil at 24 000 rpm for 1 min at ambient temperature. Subsequently, 50 μL of the emulsified solution was diluted 100 times with 0.1% SDS solution. Absorbance of the diluted emulsion was evaluated at 500 nm using Evolution 220 (Thermo Fisher, Waltham, MA, U.S.A.). The emulsifying activity index (EAI) was calculated as follows:

of OGP as a surfactant to form AOPO-in-water nanoemulsions. The pH, thermal properties, and storage stabilities of the nanoemulsions were also investigated.

2. EXPERIMENTAL SECTION 2.1. Materials. Ovalbumin powder (82%) was supplied by Sigma Chemical Co. (St. Louis, MO, U.S.A.). D-Lactose monohydrate (98%) was obtained from Tokyo Chemical Industry Co., Ltd. Q Sepharose fast flow (FF) ion-exchange packing was supplied by Shiyuanye Co., Ltd. (Shanghai, China). Prestained protein standard markers and polyacrylamide gels were procured from Bio-Rad Laboratories (Hercules, CA, U.S.A.). AOPO was a present from South China Agricultural University. Fluorescein isothiocyanate (FITC) and Nile red were obtained from Sigma-Aldrich. All of the experiments involved deionized (DI) water. All of the chemicals were analytical-grade reagents. 2.2. Preparation of Ovalbumin−Lactose (Ova−Lac) Conjugates. This method originated from Liu and Zhong,23 with a slight modification. Ovalbumin and lactose with a mass ratio of 1:1 were hydrated for 6 h in distilled water, and the dispersion pH was modified to7.0 using 0.1 N HCl and NaOH. The well-dispersed solution was subsequently lyophilized (ALPHA 2-4 LDplus, Christ Corp., Germany). The lyophilized powder was incubated for 6, 12, 24, and 48 h at 60 °C and 79% RH.6,24 2.3. Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis (SDS−PAGE). The SDS−PAGE of the samples was performed according to a previous study, with slight modifications.23 A precast 12% gradient polyacrylamide gel was used in the reducing SDS−PAGE experiment. The protein sample solution (5 mg/mL) was diluted 5 times in a SDS−PAGE sample buffer (Bio-Rad Laboratories, Hercules, CA, U.S.A.) and heated with a water bath at 95 °C for 5 min. The samples (10 μL each) were injected into the gel lane, and electrophoresis was conducted at 200 V. When the indicator dye reached the gel bottom, the piece of gel was stained with Coomassie Brilliant Blue G-250 and destained until satisfactory bands were visible. The gel was scanned using Gel Doc EZ documentation system (BioRad Laboratories). 2.4. Characterization of the Ova−Lac Conjugates. 2.4.1. Determination of the Free Amino Groups. The ratio of the free amino groups in the Ova−Lac conjugates was measured according to the literature6 and described using trinitrobenzene sulfonate (TNBS), a specific reagent for amino groups. This reagent reacts with free amino groups to yield trinitrophenyl derivatives under mild conditions. Furthermore, 1 mL of ovalbumin or Ova−Lac solution (0.5 mg/mL) was mixed with 1 mL of 0.1% TNBS and 1 mL of 4% sodium bicarbonate (NaHCO3) at pH 8.5. The mixture was incubated at 40 °C for 2 h. Afterward, 1 mL of 10% SDS was added to disperse the protein by adding 0.5 mL of 1 M HCl (Evolution 220, Thermo Fisher, Waltham, MA, U.S.A.), and the absorbance of the dispersion was evaluated at 344 nm. 2.4.2. Fluorescence Spectroscopy. Ovalbumin and Ova−Lac conjugate dispersions (0.1 mg/mL) were prepared with DI water. A quartz cuvette (1 cm) was used at 25 °C. The excitation wavelength was 280 nm, and the emission wavelength ranged from 300 to 480 nm. The slit width of excitation and emission was 5 nm.25 Fluorescence spectra were recorded using a Hitachi fluorescence spectrophotometer (F-4600, Hitachi, Japan). 2.5. Functional Properties. 2.5.1. Thermal Stability. Protein samples were dispersed in DI water (5 mg/mL). pH of the dispersion was then modified to 2−7.0 with 0.1 N HCl and NaOH. The dispersions sealed in 10 mL glass vials were incubated in a water bath at 80 °C for 5 min. A picture of glass vials was taken after heat treatment. The absorption of the dispersions was recorded at a wavelength of 280 nm before and after the heat treatment. 2.5.2. Emulsifying Property. The emulsifying activity of the protein samples was calculated on the basis of a previous study, with slight modifications.26 The samples were dispersed in a pH 7.0 phosphate buffer at a protein concentration of 5 mg/mL using a T-18 high-speed homogenizer (IKA, Germany). About 8 mL of protein dispersion was

EAI = (2.303 × 2OD500 )/(C ΦD)

(m 2 /g × 10−5)

(1)

where C represents the concentration of the protein solution, Φ is the volume fraction of oil, and D represents the path length of the cuvette. 2.5.3. Determination of the Protein Adsorption Amount on the Oil−Water Interface. The amount of absorbed ovalbumin and Ova− Lac on the oil−water interface was calculated indirectly on the basis of the amount of protein concentration in the water phase of the emulsions in accordance with previously described methods,27 with minor modifications. Briefly, ovalbumin or Ova−Lac was dispersed in sodium phosphate buffer solution with pH 4.0. The crude emulsions were formed by mixing 1% (w/w) protein solutions and 4% (w/w) sunflower oil. The mixture was subsequently homogenized at 20 000 rpm for 1 min with an IKA T-18 homogenizer. The fine O/W emulsion was fabricated by homogenizing the crude emulsion via a high-pressure homogenizer (AH-2010, ATS Industrial System, Canada) at 1000 bar and 2 passes. Sodium azide (0.022%, w/v) was added to prevent microbial growth in the solution. Freshly prepared emulsions were centrifuged at 10000g for 20 min at 4 °C. After centrifugation was performed, the cream oil phase was at the top, the protein precipitates were at the bottom, and the water phase was in the middle. The amount of non-absorbed protein in the water phase was measured using a Bradford protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). The precipitate was weighed after lyophilization. The absorption rate of protein on the oil−water interface (Cow) was calculated using the following formula: Cow = (C total − C protein − C precipitates)/C total × 100%

(2)

where Ctotal is the initial protein concentration (mg/mL), Cprotein is the protein concentration in the water phase after centrifugation (mg/ mL), and Cprecipitate is the precipitate concentration per unit volume (mg/mL). 2.6. Optimization of the High-Pressure Homogenization Condition. Each ovalbumin sample was dissolved in water (1%, w/w) with stirring at 400 rpm for 4 h at 25 °C. Subsequently, pH was adjusted to 5.0 with NaOH (0.1 M) or HCl (0.1 M). AOPO was added to the solution with a mass ratio of 5%. In this procedure, sodium azide (0.02%, w/v) was added to prevent microbial growth in the solution. This mixed dispersion was prehomogenized (IKA T-18) for 1 min at 22 000 rpm and homogenized with a high-pressure homogenizer (AH-2010) at 500, 800, and 1000 psi for 2−10 passes. Each sample was prepared in triplicates. 2.7. Optimization of the Emulsion Formulation. Emulsions with different water and oil phases were initially produced by mixing a gradient concentration of ovalbumin (1%−5%) in water with oil of various proportions (1−10%). This mixed dispersion was prehomogenized with an IKA T-18 for 1 min at 22 000 rpm and homogenized with AH-2010 at the optimum pressure determined from the above experiment. After homogenization, the sample was diluted in water to determine the particle size. 2.8. Confocal Laser Scanning Microscopy (CLSM) Experiments. The CLSM experiments of the samples were carried out according to a previous study, with slight modifications.21 FITClabeled ovalbumin and conjugated samples were obtained by mixing the FITC acetone solution (5 mg/mL) with the ovalbumin solution (10 mg/mL, pH 5.0) under mild stirring. The weight ratio of ovalbumin or conjugated samples to FITC was 20. The dispersion was set in the dark at ambient temperature overnight. Then, the dispersion was dialyzed in the dark to remove uncombined FITC using 0.01 mol/ L phosphate buffer (pH 5.0). The double fluorescence-labeled AOPO fine emulsion was produced as follows: Nile red solution was mixed with AOPO at a weight ratio of 1:20. The FITC and Nile red-labeled emulsions were B

DOI: 10.1021/acs.jafc.8b00423 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry prepared by emulsifying a combination of FITC with protein dispersion and Nile red AOPO with 5% oil volume fraction. The unembedded Nile red was also dialyzed until no fluorescence was estimated in the dialysate. The droplets were observed on CLSM (Olympus FV1200) for FITC and Nile red. The excitation wavelengths were set at 488 and 543 nm, respectively. 2.9. Stability of Emulsion against pH, Heat, and Ionic Strength. The freshly prepared fine emulsions were stored in amber glass vials and set in the dark at 25 °C for approximately 8 weeks. The emulsions (in a 20 mL amber glass vial) were incubated at 80 °C for 5 min and immediately cooled to 25 °C. The aqueous NaCl solution was mixed with the emulsion to reach the NaCl concentrations of 0.01, 0.02, 0.05, 0.10, and 0.15 M. The pH of the freshly made emulsions was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0. The droplet size distribution and polydispersity coefficient of the emulsion droplets were estimated under 25 °C using dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, U.K.). 2.10. Statistical Analysis. Results were presented as the mean ± standard deviation in tables and figures, unless stated otherwise. Data between ovalbumin and Ova−Lac glycated for different durations were compared to SPSS 17.0 to perform analysis of variance (ANOVA) tests. Significant differences were indicated by different alphabet groupings in the tables and figures, unless stated otherwise. The means were compared using a least significant difference test between the samples with a 95% confidence interval.

3. RESULTS AND DISCUSSION 3.1. Evidence for Glycation. Maillard reactions can increase the molecular weights of proteins.7 As shown in

Figure 2. Free amino group fluorescent intensity: (A) percentage of free amino groups in ovalbumin significantly (p < 0.05) decreasing over time following incubation with lactose and (B) fluorescence spectra showing more significant (p < 0.05) reduction in fluorescent intensity for Ova−Lac conjugates glycated for 12, 24, and 48 h than that of ovalbumin alone and the ovalbumin/lactose mixture (Ova−Lac glycated for 0 h).

significantly (p < 0.05) by 19.6% with lactose, whereas no significant change occurred without lactose. Ovalbumin and Ova−Lac conjugates were evaluated for conformational change around tryptophan (Trp) in ovalbumin following glycation with lactose. Tyrosine (Tyr), Trp, phenylalanine residues, and other aromatic amino acids emitted intrinsic fluorescence.28 Trp mainly contributed to the intrinsic fluorescence. Figure 2B demonstrates that the fluorescence intensity of ovalbumin glycated with lactose for 12, 24, and 48 h decreased by approximately 31, 33, and 39%, respectively, relative to that of ovalbumin alone and the ovalbumin/lactose mixture (Ova−Lac glycated for 0 h). This decrease may be due to the formation of the lactose molecular layer around the protein, which reduced the detectable autofluorescence. 3.2. Functional Property. 3.2.1. Emulsifying Property. The emulsifying property of the Ova−Lac conjugate is illustrated in Figure 3A. The incubation time of 12 h significantly enhanced the emulsifying ability of the conjugate (p < 0.05). Conversely, a long time of 24−48 h negatively affected the emulsification property. The enhanced hydrophilic capacity of ovalbumin was caused by the glycation with lactose, with the condensation between hydrophilic lactose groups and amino groups.1 This enhanced capacity may have contributed to the increased emulsifying property of ovalbumin. An increased emulsifying capacity of glycated soy protein isolate

Figure 1. SDS−PAGE profile of ovalbumin before and after glycation. Molecular weights of Ova−Lac glycated for 12, 24, and 48 h increased to 48.3, 48.4, and 48.8 kDa, respectively, compared to that for 0 h or ovalbumin alone at 45 kDa.

Figure 1, the SDS−PAGE profile confirmed the glycation of ovalbumin and lactose via the Maillard reaction following incubation at 12, 24, and 48 h. The ovalbumin molecular weight is 45 kDa,13 which can be ascribed to the strong band in lane 2. When heated with lactose (lanes 3−5) for 12, 24, and 48 h, the bands increased to 48.3, 48.4, and 48.8 kDa, respectively. These results suggested that high-molecular-weight compounds were developed in comparison to ovalbumin incubated in the absence of lactose for 48 h, and its molecular weight still remained at 45 kDa. Evidence of the Maillard reaction between ovalbumin and lactose was also confirmed by the data of the free amino groups present in ovalbumin and the conjugates. Figure 2A illustrates that the free amino groups of the heated ovalbumin decreased C


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Figure 3. Emulsifying properties and emulsion droplet size of Ova− Lac conjugates: (A) emulsifying property of Ova−Lac glycated for 0, 12, 24, and 48 h and (B) emulsion droplet size distribution of Ova− Lac glycated for 0, 12, 24, and 48 h. EAI = emulsifying activity index.

Figure 4. Thermal properties of Ova−Lac conjugates: (A) representative photographs of aqueous dispersion of 5% (w/v) Ova−Lac glycated for 24 h before and after heating at 80 °C for 5 min and (B) absorbance of aqueous dispersion consisting of 5% (w/v) Ova−Lac glycated for 0 and 24 h before and after heating at 80 °C for 5 min.

with soy-soluble polysaccharide was reported at a low temperature of 25 °C with a RH of 75% for 36−96 h.29 The emulsion droplet size distribution is shown in Figure 3B. Glycation for 24 and 48 h generated an unstable suspension of large-sized droplets (about 1.1 μm). Figure 3B also illustrates the increase in the droplet size of Ova−Lac conjugates with an increased incubation time. The conjugates of 48 h of incubation showed the largest droplet size with the worst emulsifying capacity possibly as a result of the thermal aggregation of ovalbumin during glycation, which resulted in decreased solubility and folded structure. 3.2.2. Thermal Properties. Ovalbumin loses its molecular stability and aggregates when heated.30 The thermal stabilities of ovalbumin and Ova−Lac dispersion were evaluated before and after thermal treatment at 80 °C for 5 min (Figure 4A). Before the aqueous solution was heated, ovalbumin and Ova− Lac conjugates were transparent at pH 2.0−7.0 (Figure 4A). After the heat treatment, the dispersions of ovalbumin without glycation remained transparent at pH 2.0, 3.0, and 4.0 and became turbid at pH 5.0 and 6.0, while that of the glycated ovalbumin remained relatively transparent at pH 5.0 and 6.0. This phenomenon was consistent with a previous study on the thermal aggregation properties of glycated whey protein,16 which can be ascribed to thermal denaturation stability improvement caused by modification. Notably, the turbidity of ovalbumin and Ova−Lac was also pH-dependent, as shown in Figure 4B. The protein solution became turbid when the solution pH was approximately 5.0 and the isoelectric point of ovalbumin was 4.8. Consequently, when the solution pH was below 5.0, ovalbumin and Ova−Lac

Figure 5. Absorption amount of ovalbumin and Ova−Lac on the oil− water interface. The absorption of Ova−Lac glycated for 12 h (p < 0.05) significantly increased in comparison to that of the ovalbumin control. A prolonged glycation time attenuated this increased absorption slightly.

carried many positive charges to increase the repulsion between protein molecules, which maintained the transparency of solution. When the solution pH was greater than 5.0, ovalbumin and Ova−Lac were highly negatively charged to increase the repulsion and maintain the transparency of solution. Moreover, Figure 4B shows that, after glycation for 24 h, the Ova−Lac dispersion became transparent at all pH values, except for a little turbidity at pH 5.0, as depicted in D


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Figure 6. Effects of the homogenization pressure level and number of passes on the droplet size of the emulsion, which was composed of 5% oil, 1% protein, and 94% water. The optimal conditions to obtain nanoemulsions near 200 nm were at 1000 bar and 8−10 passes.

Figure 8. CLSM image of Ova−Lac AOPO emulsion: (A) representative image from the 5% oil phase containing Nile red and (B) FITC-labeled Ova−Lac conjugates. AOPO, aged orange peel oil; CLSM, confocal laser scanning microscopy. Figure 7. Mean droplet size of varying protein/oil ratios under highpressure homogenization. The optimal ovalbumin amount was 1−3%, and that of oil was ≤6%.

glycation resulting in ovalbumin unfolding, reduced surface availability at the oil−water interface, and partial lost solubility. These results were consistent with those illustrated in Figure 3B, where a long glycation time caused a large droplet size, decreased solubility, and increased structural folding. The adsorption of bovine serum albumin, lysozyme, and insulin at the oil−water interface is related to the protein concentration and molecular weight.31 3.3. Optimum High-Pressure Homogenization Condition. The proteins and oil were refined and uniformly mixed during high-pressure homogenization. Protein molecules were drastically sheared and collided that they became absorbed on the surface of the oil phase; they subsequently dispersed into small particles and finally formed the oil−water emulsion. The nanosized emulsion production by high-pressure processing was optimized. The process parameters of the formulation, which comprised 5% oil, 1% protein, and 94% water, were screened. This emulsion was finally processed by high-pressure homogenization at three increasing pressure levels (500, 800, and 1000 bar) and 10 passes. The mean droplet size of the emulsion was also evaluated, as shown in Figure 6. The final nanoemulsion product was successfully prepared when the average particle size of approximately 200 nm was obtained. The results showed that this size was achieved with 8 or 10 passes at 1000 bar. The maximum pressure for the homogenizer was 1000 bar. Therefore, homogenization can only be conducted at a maximum of 1000 bar. At 500 or 800 bar, the minimum mean particle sizes were not lower than 250 nm, even after 10 passes. According to these findings, the processing parameters were set and remained at 1000 bar with

Table 1. Z Diameter of the Nanoemulsiona AOPO (%)

Ova (%)

water (%)

4 5 6 4 5 6

1 1 1 2 2 2

95 94 93 94 93 92

Ova-based (nm)

PDI

± ± ± ± ± ±

0.267 0.288 0.347 0.211 0.256 0.311

254 331 382 329 362 418

18 12 19 20 25 27

Ova−Lac-based conjugates (nm)

PDI

± ± ± ± ± ±

0.106 0.083 0.223 0.251 0.268 0.311

192 210 311 308 351 397

11 10 25 22 19 15

a AOPO, aged orange peel oil; Ova, ovalbumin; Ova−Lac, ovalbumin− lactose; and PDI, polydispersity index.

Figure 4A. The heat stability was increased possibly as a result of the saccharide groups found in the conjugated protein, which hindered the thermal aggregation and weakened the heat absorption of the peptide chain. Cow values of ovalbumin and Ova−Lac glycated for different durations are shown in Figure 5. The absorbed amount of ovalbumin without glycation was 16.13%, which significantly increased to 23.31% after glycation for 12 h (p < 0.05). This result may be caused by the increased hydrophilic capacity of Ova−Lac, which improved the adsorption of protein at the interface betweent oil and water. However, the absorption amounts of conjugates at 24 and 48 h were lowered by 2.75 and 5.12%, respectively. This phenomenon can be due to the excess E


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Figure 9. Droplet sizes of ovalbumin and Ova−Lac AOPO emulsions under different treatments: (A) heating at 50, 60, 70, and 80 °C, (B) pH 2.0− 7.0, (C) NaCl concentration ranging from 0.01 to 0.15 mol/L, and (D) after 8 weeks of storage at 25 °C. Ova−Lac AOPO emulsions remained more stable despite changes in the temperature, pH, isotonic strength, and long-term storage than those with ovalbumin (p < 0.05). AOPO = aged orange peel oil.

3.5. Emulsion Structure. To demonstrate that ovalbumin was located at the oil and water interface and to show that the structure of the emulsion was an O/W type, we used FITClabeled glycated ovalbumin for emulsion synthesis and used Nile-red-labeled AOPO as the oil phase. The AOPOencapsulated emulsion was prepared with 5% (w/w) AOPO and 1% (w/w) protein. The pH of the fine emulsion was adjusted to 5.0 to enhance hydrophobicity and electrostatic repulsion, which allowed for the observation of droplets on a micrometer scale through CLSM. A CLSM image at 100× magnification (Figure 8) was selected. This image shows considerably small particles from the Nile-red-labeled oil and dual-fluorescence-labeled droplets. The red fluorescence droplets in Figure 8A indicated that the oil particles were nanosized and homodispersed in the water phase. Figure 8B illustrates the droplets with a red fluorescence ball and an outer ring with green fluorescence, which confirmed that glycated ovalbumin was found at the interface of oil and water. Therefore, the glycated ovalbumin-based AOPO emulsion particles are nanosized and have an oil−water structure. 3.6. Effects of Chemical and Physical Treatment on Emulsion Stability. Heating was conducted from 50 to 80 °C at 10 °C intervals for 5 min with the final emulsion at pH 7.0 to simulate pasteurization (Figure 9A). The heating results showed that the emulsion particle size of the Ova−Lac-based emulsion did not significantly increase (p > 0.05). In ovalbumin-based emulsion, the droplet size parameters were remarkably increased (p < 0.05), indicating that glycated ovalbumin restrained the flocculation of the emulsion droplets. Protein molecules in the emulsion were combined in the AOPO and water interphase, and other molecules were in the water-soluble phase. The absence of droplet flocculation

8 homogenization passes. The high-pressure homogenization resulted in considerably well-preserved fine emulsions having a narrow distribution of particle sizes, thereby improving the kinetic stability of the systems. Consequently, the stable region can be further widened to reduce the amount of protein for kinetically stable formulations. 3.4. Optimization of the Emulsion Formulation. Most hydrophobic amino acids of ovalbumin are hidden within the molecules instead of being exposed to the molecular surface when ovalbumin is in the acidic or neutral condition. Nevertheless, the emulsification ability of ovalbumin is relatively strong at pH 5.0, and its molecules exhibit high hydrophobicity and fluidity.32,33 Figure 7 shows how the protein/oil ratios influenced the non-creamy formulations. Notably, stable nanoformulations were obtained when the ovalbumin percentage was between 1 and 3% and the oil percentage was 6%. The measured sizes evidently depended upon protein ratios. In particular, when a high protein proportion was used, the droplet sizes were remarkably larger than the nanosize. However, if a low protein proportion was used, the droplets showed small sizes and surface coverage. Nevertheless, in terms of the mean droplet sizes (Table 1), the key step in evaluating the particle size may be represented by the amount of protein adsorbed optimally in the oil and water interface during high-pressure homogenization. When emulsion droplets were formed with an unequivalent interfacial adsorption, they rapidly gathered to aggregate large particles, unless a balanced surface coverage occurred. Consequently, if the formulation was rich in protein, then the final droplet sizes were mainly dependent upon the vaild energy injected to the emulsion. F


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implied that the protein/lactose conjugation in the interphase between AOPO and water prevented macrographic aggregation after the protein denaturalized at a high pressure. The Ova−Lac-based emulsions at pH 2−7 were more homogeneous than the ovalbumin-based emulsions. However, the droplet size suddenly increased at pH 4.0 and subsequently reached the maximum level at pH 5.0, which was within the isoelectric point of ovalbumin as a result of its loss of electrostatic repulsion (Figure 9B). Serial dilutions of salt (NaCl) solution added to the emulsion (Figure 9C) showed that the droplet sizes of ovalbumin-based emulsions increased more rapidly with the increased NaCl concentration than those of Ova−Lac-based emulsions. The ovalbumin-based emulsions containing more than 0.02 mol/L NaCl exhibited creaming and phase separation, whereas the Ova−Lac-based emulsions under the same conditions were visually homogeneous. After approximately 50 days of storage under ambient conditions, the particle sizes of the ovalbumin-based emulsions continued to enlarge (Figure 9D). The difference between the Ova−Lac- and ovalbumin-based emulsions can be ascribed to the glycated ovalbumin molecular layer, which was stable under adverse physical and chemical conditions, as also concluded in previous studies.34,35 Heat and NaCl molecules can lead to protein aggregation. Consequently, the emulsion droplets coalesce. On the contrary, the lactose layer glycated onto ovalbumin and hindered the coalescence of droplets. During storage, the oil−water interface was stable; therefore, the lactose layer on the droplet surface prevented the aggregation of emulsion particles. In conclusion, ovalbumin was successfully glycated with Dlactose through Maillard reaction via dry heating. Covalent linkage was verified through SDS−PAGE and free amino group content analyses. The improved emulsifying capacity and thermal stability of Ova−Lac conjugates indicated that they can be used as a functional emulsifier and stabilizer, especially near the isoelectric point. Therefore, the glycated ovalbuminbased AOPO emulsion particles are nanosized and stable against variations in the temperature, pH, ionic strength, and long-term storage. The particle structures are of O/W type.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Gang Liu: 0000-0002-1610-6729 Funding

This study was supported by the National Natural Science Foundation of China (31771925 and 31401640). Notes

The authors declare no competing financial interest.

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REFERENCES

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Maillard-Reaction-Functionalized Egg Ovalbumin Stabilizes Oil

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