Substantial Enhancement of the Antioxidant Capacity of an α-Linolenic

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

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Substantial Enhancement of the Antioxidant Capacity of an α‑Linolenic Acid Loaded Microemulsion: Chemical Manipulation of the Oil−Water Interface by Carbon Dots and Its Potential Application Mengna Hou, Qing Li, Xiaoxue Liu, Chao Lu, Sen Li, Zhanzhong Wang,* and Leping Dang*

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School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People’s Republic of China S Supporting Information *

ABSTRACT: Various active ingredients play a crucial role in providing and supplementing the nutritional requirements of organisms. In this work, we attempted to chemically manipulate the interfacial microstructure of oil−water microemulsions (ME) with carbon dots (CDs), concentrating on substantially enhancing the antioxidant capacity of α-linolenic acid (ALA). To this end, CDs were synthesized and introduced into an ME. The molecular interaction of surfactant with CDs was investigated by Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR). The microstructure of the ME was monitored by transmission electron microscopy (TEM) and cryo-electron microscopy (cryo-EM). The cryo-EM result showed the oil−water interface in the ME was better defined after the CDs were loaded, and 1H NMR proved the CDs were distributed mainly at the interface. On the basis of these results, interfacial models were proposed. Final evaluation results demonstrated the stabilizing effect and oxidation-inhibition ability of the ALA-loaded ME was substantially enhanced after the introduction of the CDs, indicating a “turn off” effect of the interface. Interestingly, CDs do not affect the in vitro release of ALA, indicating a “turn on” effect of the interface. This work provided a successful interface manipulation with a nanocarrier that can be used for a large diversity of food nutraceuticals. KEYWORDS: microemulsion, antioxidant capacity, interfacial microstructure, chemical manipulation, potential application

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oil-in-water ME to load curcuminoid.9 The results indicated that the relative bioavailability of curcuminoid loaded in the ME was enhanced an average of 9.6-fold over that of a curcuminoid suspension. Wang et al. investigated the antioxidant stability of α-linolenic acid loaded (ALA-loaded) oil-in-water ME. It was found that the antioxidant capacity of α-linolenic acid in ME was enhanced by about 80% compared with that of ALA in an oil solution.10 As shown in Scheme 1, the interface between the two phases, working as a separating wall, plays a significant role in protecting biologically active molecules from undesired damage.11 The interface of the ME, with uniform nanosized droplets and a double-layered structure, can dramatically affect the interaction between the active ingredients in the inner phase and the unfavorable factors in the outer phase, such as oxygen, which can alter the function of the active ingredients during delivery as a result of an oxygenation reaction.9,10 Therefore, the microstructure of such an interface requires exhaustive investigations. Generally, it is suggested that surfactant molecules, having “head” parts containing polar or ionic functional groups and “tail” parts containing hydrocarbon chains, are located at the oil−water interface as a monolayer surrounding the oil core and thus stabilizing the ME.12,13 Over the past few years, several techniques have been developed, such as forward recoil

INTRODUCTION The intensive research in all fields of micro- and nanoscale science has generated a variety of approaches for the engineering of microparticles and nanoparticles with tailored properties.1,2 Microemulsions (MEs), which function as membrane-mimetic systems,3,4 have attracted much attention, as their properties are widely acknowledged as being more advantageous than those of conventional host media like dextrin5,6 and because they have great potentials in a variety of applications, from delivery nanocarriers that protect various nutraceuticals to nanoscale reactors for improving the efficacy of enzymatic and chemical reactions.7,8 An ME with a doublelayered and compartmentalized structure is schematically illustrated in Scheme 1. Ping et al. utilized a well-constructed Scheme 1. Schematic Representation of the O/W ME with a Double-Layered and Compartmentalized Structure

Received: April 17, 2018 Revised: June 5, 2018 Accepted: June 13, 2018

© XXXX American Chemical Society

A

DOI: 10.1021/acs.jafc.8b01991 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry spectroscopy,14 dynamic secondary-ion mass spectroscopy,15 neutron scattering, and neutron reflectivity16 to quantify the concentration profiles at buried interfaces, such as those in MEs. However, each technique requires the selective labeling of one of the components, and none can simultaneously provide information related to the microstructure within an interfacial region, which is important for the final properties of a material.17 The poor or inadequate investigation of the interface leads to mistaken interpretations of the interface microstructure, thus restricting the applications of MEs as highly efficient delivery systems. Carbon dots (CDs) of 2−10 nm in diameter are frequently used as nanoprobes in the fields of drug delivery18 and biological sensing19 and in related research.20,21 CDs are usually functionalized with some polar groups, such as ammonium or carboxyl groups.22,23 Surface-functionalized CDs have gained enormous significance in materials science because of their unique physicochemical properties.24−27 The highlighting of the interface of MEs using CDs will be of great interest. This is a very attractive strategy because of the amphiphilic character of surfactant molecules,28−30 whose polar groups interact strongly with CDs, resulting in the distribution of the CDs at the oil−water interface. A hypothesis has been suggested that the microstructure of the interface could be adjusted in the aqueous media, and as consequence, the field of application of ME could be changed accordingly, especially where there is a strong interdependency between the microstructure of the interface and the applications of the ME. In this contribution, an oil in water (O/W) ME containing a hydrophobic active component (ALA) was used as a host for loading CDs. In this self-aggregated homogeneous system, the hydrophobic active components, ALA, were located in the hydrophobic pool, whereas the CDs were placed in the water pool in close intimacy with the interface, thus generating a strong protective barrier for ALA molecules against oxidation. Hence, to prove the great gain brought by CDs prepared by a microwave method from citric acid and polyethylenimine, the CDs were introduced into the outer water phase of the ME in order to investigate the interfacial microstructure of the ME and then manipulate the properties at the nanoscale. The prepared CDs were characterized in relation to their morphology, size, and surface chemical composition. The surfactant−CD interaction at the interface was confirmed by Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR), whereas the microstructural morphology of ME was evidenced by transmission electron microscopy (TEM) and cryo-electron microscopy (cryo-EM). Finally, the antioxidant capacity, the stability against the environmental stress factors (e.g., temperature, pH, and salinity), and the ability of the CD−ALA-loaded ME to release ALA in vitro were also studied.

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Company Ltd. All the syntheses were performed in double-distilled water. Preparation and Characterizations of Carbon Dots (CDs). The microwave pyrolysis approach was adopted to synthesize the CDs according to the literature with some improvements.31,33 First, 3 g of citric acid (as the carbon source) and 1 g of polyethylenimine (PEI, as the passivator) with a weight ratio of 1:3 were added into 50 mL of double-distilled water; then, the mixture was heated in a 500 W microwave oven for 10 min. The solution changed from colorless to yellow, which indicated the formation of PEI-modified CDs. Then, the yellow solution was freeze-dried and stored at −20 °C for the following experiments. An aqueous solution of CDs (5 μL with a concentration of 5 mg/mL) was added onto a 400-mesh carboncoated copper grid and then dried under vacuum at 40 °C. The particle size and morphology of the CDs was determined by fieldemission transmission electron microscopy (Tecnai G2 F20 S-TWIN (200 kV), FEI). The TEM samples were prepared by dropping the samples onto a copper grid and drying them in air. The particle-size distribution of the CDs was measured at 25 °C using a Nano Zetasizer based on dynamic light scattering (DLS; Zetasizer Nano ZS90, Malvern Instruments Ltd.). Then, the surface groups of the CDs were confirmed by analyzing Fourier-transform infrared (FTIR) spectra (NICOLET 8700, Thermo Nicolet Corporation). Preparation of CD−ALA-Loaded O/W ME. Isoamyl acetate, polyoxyethylene castor oil EL (CrEL), and ethanol were used as the oil phase, surfactant, and cosurfactant, respectively, to prepare the ME. The ME was prepared according to our previous study.32 On the basis of that, various concentrations of CDs (i.e., 1, 3, 5, 6, 8, and 10 mg/mL) were dissolved into the water phase to form CD-loaded MEs. The maximum CD concentration in the ME was established by comparing the visual appearances of MEs with different CD concentrations. Similarly, ALA at concentrations of 15, 20, 25, 30, 35, 40, and 45% (w/w) was loaded in the ME. The maximum capacity of ALA was determined on the basis of ultraviolet spectrophotometry (UV-3300, Meipuda Instrument Company, Ltd.) at 320 nm. In light of these findings, CD concentrations (0.025, 0.05, 0.1, 1, 2, 3, 4, and 5 mg/mL) below the maximum were chosen to form CD-loaded MEs for the following experiments. Meanwhile, an ALA concentration of 30% (m/m) was established to prepare the CD−ALA-loaded MEs. In the following investigations, the original ALA concentration was fixed at 30% (m/m) without additional information. Characterization of CD-Loaded O/W ME. First, the effects of CD concentration on the particle size and interfacial tension of the ME were determined. The particle sizes of the CD-loaded MEs were determined using DLS, and the microstructural morphology was investigated by transmission electron microscopy (TEM). TEM samples were prepared by depositing a drop of a diluted microemulsion sample onto a film-coated copper grid, then staining it with a drop of a 2% aqueous solution of phosphotungstic acid, and allowing it to dry at room temperature before examination. The interfacial tension for CD-loaded MEs was determined by the BZY-A method using an automatic interface tensiometer (QBZY, Shanghai Fangrui Instrument Company, Ltd.). Then, with a fixed CD concentration of 2 mg/mL, an FTIR spectrum of a CD-loaded ME was registered in order to observe the changes in the functional groups of the CDs involved in the interaction with the surfactant at the interface. The microstructure of the oil−water interface was probed using cryo-electron microscopy (cryo-EM) based on a JEM1200EX (120 kV) electron microscope and an FEI Titan Krios (300 kV) microscope. Samples of MEs and CD−MEs (about 5 μL) were dripped several times onto the treated copper net, and the excess solution was quickly absorbed with filter paper. After quick-freezing, the sample was observed at −173 °C. 1 H NMR Measurement. In this part, a CD concentration of 2 mg/mL was chose to form the CD-loaded ME and CD−ALA-loaded ME (i.e., an ME with CDs in water phase and ALA in the oil phase). The 1H NMR measurements for ME, the CD-loaded ME, the ALAloaded ME, and the CD−ALA-loaded ME were performed using a Varian Inova spectrometer (500 MHz). Deuteroxide, instead of double-distilled water, was used when preparing the MEs.

MATERIALS AND METHODS

Materials. Citric acid (>99.5%) was purchased from Tianjin Bodi Chemical Company Ltd. Polyethylenimine (AR grade) was obtained from Shanghai Maclin Biochemical Technology Company Ltd. Isoamyl acetate (AR grade) was purchased from Tianjin Chemical Reagent Supply and Marketing Company Ltd. Polyoxyethylene castor oil EL (CrEL) was purchased from Shanghai Source Leaf Biotechnology Company Ltd. Tween 80 was bought from Beijing Solarbio Science and Technology Company, Ltd. ALA (99%) was purchased from Sigma-Aldrich Company LLC. Ethanol was purchased from Tianjin Rionlon Bohua Pharmaceutical Chemical B

DOI: 10.1021/acs.jafc.8b01991 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Antioxidant-Capacity Determination of CD−ALA-Loaded ME under Different CD Concentrations. The antioxidant capacities of CD−ALA-loaded MEs were assessed by the amount of ALA loss. CD−ALA-loaded MEs with different CD concentrations, prepared in the Preparation of CD−ALA-Loaded O/W ME section, were investigated in this experiment. These MEs were kept at room temperature for 15 days. The ALA concentrations in these samples were determined daily with a UV3300 at 320 nm, and the loss ratios of ALA were compared on the basis of the following formula:

loss ratio of ALA (%) = (Coriginal − C later)/Coriginal

(1)

where Coriginal refers to the ALA concentration of the original sample, and Clater refers to the ALA concentration of the sample after it was stored for some days. Antioxidant-Stability Determination of CD−ALA-Loaded MEs under Environmental Stress Factors. In this part, the CD concentration was fixed at 2 mg/mL when the CD−ALA-loaded ME was formed. The effects of storage temperature, salinity, and pH on the antioxidant stability of the CD−ALA-loaded ME and ALA-loaded ME were investigated. The investigated temperatures were 25 °C (room temperature), 37 °C (body temperature), and 50 °C (processing temperature), and ALA concentration was determined after storage for 20 days at each temperature. To study the effects of pH and salinity, phosphate buffers of pH 2.0 (artificial stomach) and pH 7.0 (enteral environment) and NaCl solutions with concentrations of 0.9% (physiological saline solution) and 4.5% (high-salt solution), both containing CDs, were used as the water phases. Then, the ALA concentration was estimated after 20 days of storage. Similarly, the effects on ALA-loaded MEs were determined in the same conditions as a contrast for the CD−ALA-loaded MEs. In Vitro Release Determination of ALA from CD−ALALoaded O/W MEs. In vitro release experiments were performed in order to determine the controllability of the oil−water interface after the introduction of CDs, according to previously reported methods with slight modifications.7 A CD concentration of 2 mg/mL was adopted in this part. The release curves of ALA from CD−ALAloaded MEs were assessed by dialysis in pH 2.0 (gastric acid environment) and pH 7.0 (intestinal tract environment) buffers (PBS) containing 0.1% (w/v) Tween 80. The CD−ALA-loaded MEs were placed in the dialysis tubes (MWCO 3.5 kDa). Afterward, the dialysis tubes were introduced in each buffer (200 mL), incubated in a shaking bath at 37 °C, and stored for different times under stirring, with an agitation speed of 100 rpm. Aliquots of the mixture (2.0 mL) were withdrawn at specific times (0.5, 1, 1.5, 2, 5, and 8 h), and an equivalent volume of fresh buffer was added. The concentration of ALA in the release medium was determined by a UV3300 at 320 nm, as indicated. Statistical Analysis. Data were presented as means ± standard deviations. The data from the measurements were subjected to analysis of variance (ANOVA) using the SPSS 17.0 software package (IBM). Statistical analysis was performed using a Duncan’s multiplerange test to identify significant differences among the mean values (P < 0.05).

Figure 1. Particle-size distribution and representative TEM image of the CDs. Scale bar = 20 nm.

Figure 2. FTIR spectra for the prepared CDs and CD-loaded MEs.

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RESULTS AND DISCUSSION Morphology, Size, and Structure of the CDs. The particle-size distribution of the prepared CDs estimated using dynamic light scattering (DLS) is shown in Figure 1. The average particle size was about 3.0 nm with a polydispersity index (PDI) of 0.215. The particle size was also estimated from TEM images. A representative TEM image is inserted in Figure 1. A relatively narrow size distribution ranging from 2 to 5 nm with an average size of about 3 nm was found, which was well in agreement with that obtained by the DLS method. Furthermore, the Fourier-transform infrared (FTIR) spectrum was acquired to characterize the organic functions on the CDs. As shown in Figure 2a, a broad absorption peak at 3422 cm−1 was attributed to N−H vibrational stretching, whereas the

narrow and strong peak (1582 cm−1) corresponded to the scissoring vibration of N−H. The peaks at 1024 and 901 cm−1 were attributed to C−N vibrational stretching and out-of-plane bending vibration, respectively. These four peaks support the presence of amino groups (−NH2) on the CDs. The peak at 1391 cm−1 referred to the CO vibration of the −COOH groups. Skeletal vibration of the aromatic ring was also observed at 611 cm−1. These results are in accordance with those reported in the literature.22,33,34 Thus, −NH2 and −COOH groups are found on the CD surfaces, as schematically represented in Figure 2a. Maximal Loading Concentration of CDs and ALA in ME. The visual appearance of CD-loaded MEs with different CD concentrations of 1, 3, 5, 6, 8, and 10 mg/mL is shown in C

DOI: 10.1021/acs.jafc.8b01991 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Figure 3. As observed in Figure 3a−f, the system became opaque for concentrations of CDs exceeding 5 mg/mL,

Figure 3. (a−f) Visual appearance of MEs with different CD concentrations of 1, 3, 5, 6, 8, and 10 mg/mL, (g) visual appearance of ME, (h) ALA-loaded ME, (i) CD-loaded ME, and (j) CD−ALAloaded ME after 90 days of storage.

Figure 4. Change of the absorbance at 320 nm with ALA concentration in ALA-loaded MEs.

whereas below this value, the system was homogeneous, transparent, and stable. The PDIs of samples determined by DLS were shown in Table 1. From Table 1, the PDI is more

assessed by TEM. The corresponding images are displayed in Figure 5. As a first observation, it can be seen that the ME consists of particles that are uniformly dispersed with no aggregation and that display a spherical morphology regardless of the concentration of CDs. However, a significant effect of this parameter on the size of the particles was noticed. Therefore, if the ME prepared without CDs has particles of 26 nm on average, the size drastically decreases even with the first concentration of CDs. The particle-size distributions determined by DLS for each sample are shown in Figure 6. The corresponding average sizes summarized in Table 2 are well in agreement with those provided by TEM. These outstanding results are explained by the effect of the CDs on the intermolecular interactions in the water phase of the droplet, resulting in a tightening of the water layer, and thus smaller particles with enhanced dispersion and larger spaces between them are generated. It is worthwhile noticing that a concentration of 2 mg/mL CD is the maximum concentration at which the smallest thermodynamically stable particles are obtained (14.37 ± 0.79 nm). Indeed, further increases of the CD concentration did not change the size of the particles (Figure 6 and Table 2). The CD−surfactant interface was better visualized by performing cryo-EM. The typical images for an ME without and with 2 mg/mL CDs are shown in Figure 7a,b, respectively. It can be clearly observed that the oil and water domains are discretely separated by an interface that appears after the CDs are loaded into the ME. How the CDs favor the formation of such interfaces is the subject of the following section of this work. It is well-known that, usually, the interfacial tension is affected by temperature and pressure as a result of changing the intermolecular forces. Figure 8 displays the influence of the CD concentration on the interfacial tension. Accordingly, the surface tension at the interface decreased as the CD concentration increased up to 2 mg/mL; afterward, it started to increase again. These results show that the intermolecular forces are weak for CD concentrations lower than 2 mg/mL, which is in accord with the results obtained by TEM, in which larger spaces were observed for concentrations below 2 mg/ mL. FTIR Analysis of CD-Loaded ME. The FTIR spectrum of the CD-loaded ME is shown in Figure 2b in comparison with that of the CDs in order to observe the interaction between CDs and the surfactant at the interface. Peaks centered at 2932, 1718, 941, and 845 cm−1 were noticed and attributed to C−H stretching vibration in alkanes, CO stretching

Table 1. Appearance and Polydispersity Indexes (PDIs) of Different ME Samples CD concentration (mg/mL)

visual appearance of sample

1 3 5 6 8 10

transparent transparent transparent turbid turbid turbid

PDI 0.294 0.287 0.303 0.576 0.688 0.602

± ± ± ± ± ±

0.023 0.019 0.028 0.036 0.027 0.024

than 0.5 when the CD concentration exceeds 5 mg/mL, meaning the system is not homogeneous. On the basis of this finding, it was established that the maximum concentration of CDs that could be loaded in the ME was 5 mg/mL. An intermediate CD concentration of 2 mg/mL was chose to form the ME, and the ALA concentration was determined to be 30% of the oil-phase weight in the following investigation. The images displayed in Figure 3g−j show comparatively the visual appearance of the ME, the ALA-loaded ME, the CDloaded ME, and the CD−ALA-loaded ME, which have been stored at room temperature for 90 days. A change in color is evident after the addition of CDs, which clearly indicates that the CDs are responsible for the color intensification of the ME. To rationalize this finding, the modification of the interface microstructure affected by the CDs was taken into consideration. The concentration of ALA in the ME was also evaluated, and the maximal value was established on the basis of the UV absorbance at 320 nm. For this end, ALA-loaded ME with different ALA concentrations were prepared, and then the absorbance was measured for each sample. The maximum of the intensity was plotted as a function of the ALA concentration. The resulting curve is illustrated in Figure 4. It can be noticed that the intensity increases with the concentration up to a value of 30% (m/m), after which it remains constant, suggesting that the maximal capacity of ALA in this prepared ME is 30% (m/m). In this investigation, the concentration of ALA was set as 30% without additional explanation in the following experiments. Changes in the Physicochemical Properties of ME Influenced by CDs. The morphology and sizes of the CDloaded MEs containing different concentrations of CDs were D

DOI: 10.1021/acs.jafc.8b01991 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 5. TEM images of CD-loaded MEs with different CD concentrations: (a−f) 0, 0.025, 0.1, 1, 2, and 5 mg/mL. Scale bars = 50 nm.

Figure 7. Cryo-EM images of (a) ME (scale bar = 50 nm) and (b) CD-loaded ME (scale bar = 30 nm).

Figure 6. Size distribution of ME samples as determined by DLS with a statistical survey.

Table 2. Average Particle Sizes of CD-Loaded MEs with Different CD Concentrations CD concentration (mg/mL) 0.000 0.025 0.100 1.000 2.000 5.000

particle size of CD-loaded ME (nm) 26.33 20.64 18.59 16.42 14.37 14.29

± ± ± ± ± ±

1.32 1.01 0.97 0.86 0.79 0.72

Figure 8. Variation trend of the interfacial tensions of microemulsions with different CD concentrations.

(−COOH) groups of the CDs and the hydroxyl (−OH) groups of the surfactant molecules. 1 H NMR Analysis and Proposed Models. To further interpret the protective mechanism of CDs on ALA, 1H NMR was performed for the ME, CD-loaded ME, ALA-loaded ME, and CD−ALA-loaded ME, and the registered spectra are shown in Figure S1. Basically, 1H NMR provides information on the changes in organic molecules depending on the local chemical environment of the protons. Therefore, this analysis was performed with the aim of examining the microstructure of the surfactant−CD interface and thus establishing where the CDs are located. The corresponding chemical shifts of the

vibration in esters, and C−H bend vibration in alkenes and aromatic rings, respectively. A very intense peak at 2441 cm−1 was observed and associated with the solvent peak of D2O. The peak at 1544 cm−1 was attributed to N−H bend vibration, whereas the absorption peaks at 1466, 1345, and 1079 cm−1 arose from C−N stretching vibration, which clearly showed the existence of amides, indicating that the −NH2 groups on the CDs has been transformed. The peak at 1197 cm−1 was attributed to the −C(O)−O group of the saturated ester, which resulted from the reaction between the carboxyl E

DOI: 10.1021/acs.jafc.8b01991 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 3. Chemical Shifts of Functional Groups in the Surfactant CrEL As Determined by 1H NMR Spectroscopy σ (ppm)a

Δσ (ppm)

functional group

σA

σB

σC

σD

Δ(B − A)

Δ(C − A)

Δ(D − C)

α2-CH2 (CH2CH2O)n α1-CH2 β-CH2 (CH2)n CH3

4.156 3.646 1.931 1.529 1.236 0.853

4.149 3.636 1.926 1.521 1.229 0.846

4.165 3.641 1.916 1.525 1.232 0.849

4.145 3.633 1.919 1.517 1.224 0.841

−0.007 −0.01 −0.005 −0.008 −0.007 −0.007

0.009 −0.005 −0.015 −0.004 −0.004 −0.004

−0.02 −0.008 0.003 −0.01 −0.008 −0.008

a

A refers to ME, B refers to CD-loaded ME, C refers to ALA-loaded ME, and D refers to CD−ALA-loaded ME.

Scheme 2. Chemical Formula of the Surfactant CrEL (a) and Proposed Models for ME (b), CD-Loaded ME (c), and CD− ALA-Loaded ME (d)

be see that the oil and water domains were discretely separated after CD loading, through which the ALA in the oil phase was better protected. Effects of CD Concentration on the Antioxidant Capacity of CD−ALA-Loaded MEs. As already stated above, the ALA molecule is easily oxidized when it comes into contact with oxygen. Therefore, the main concern is to prevent this molecule from being oxygenated during the storage process. Hence, we thought that the addition of CDs to the oil−water interface would increase the barrier or turn-off effect against ALA oxidation by solidifying the interface between the oil and water domains in the ME. To demonstrate this effect, MEs with 30% (m/m) ALA containing different concentrations of CDs from 0.025 to 5 mg/mL were kept at room temperature for 15 days. After that, the concentrations of ALA were determined by UV spectrophotometry. The results are displayed in Figure 9 as loss ratio of ALA as a function of CD concentration. It is obvious that the loss ratio of ALA drastically decreased from 40 to 15% when the concentration of CDs increased from 0.05 to 0.1 mg/mL. However, the best result was obtained for the optimum concentration of CDs (2 mg/mL), with which only 5% of the ALA was lost. Above this

functional groups in the surfactants (CrEL) are listed in Table 3. It is clear that the ALA loaded in the oil phase had pronouncedly affected the α1-CH2 group (Δσ = −0.015), indicating that the location of ALA is close to the α1-CH2 group in the surfactant. Also, it is noted that the addition of CDs (Table 3, sample B) in an ME without ALA (Table 3, sample A) had a great influence on the chemical shift of the functional groups of the surfactant, especially on the (CH2CH2O)n moieties (Δσ = 0.01). This result suggests that the CDs are located at the interface of ME in close intimacy with the (CH2CH2O)n groups of the surfactant (as in Scheme 2b). Similarly, this significant effect was also observed after the addition of CDs into the ME containing ALA. However, this time, the α2-CH2 (Δσ = −0.02) and β-CH2 (Δσ = −0.01) groups were involved in the interaction. This result shows without a doubt that CDs are embedded at the interface when ALA had already been loaded in the oil phase (as in Scheme 2c). As observed in Table 2, the effect on other functional groups in the surfactant molecule was negligible. From this point of view and combined the results of FTIR, the possible microstructures of CD-loaded ME and CD−ALAloaded ME were proposed and illustrated in Scheme 2. It could F

DOI: 10.1021/acs.jafc.8b01991 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 9. Loss ratios of ALA from CD−ALA-loaded MEs with different CD concentrations stored at room temperature for 15 days.

concentration of CDs, the amount of lost ALA is practically constant, demonstrating that at this concentration, the CDs are enough to act as a protective barrier (as shown in Scheme 2c) that prevents the loss of ALA and thus enhances the antioxidant capacity of the CD−ALA-loaded ME. Moreover, as discussed above, after the addition of CDs into the ME, the thickness of the water layer decreased. Therefore, the amount of dissolved oxygen should be lower than that in an ME without CDs and thus the amount the oxygenated ALA should be accordingly lowered. Comparison of the Environmental Stability of ALALoaded ME and CD−ALA-Loaded ME. To demonstrate the great ability of CDs to strengthen the oil−water interface and to highlight the enhanced stability of these MEs upon CD addition, the effect of the storage temperature, salinity, and pH on the stability of CD−ALA-loaded ME was further investigated. To this end, the MEs were stored for 20 days at different temperatures, pH values, and ionic strength. After that, the concentration of ALA in ME was spectrophotometrically evaluated. Figure 10 illustrates the obtained results as percent of ALA loss as a function of each investigated parameter. It is expected that a low loss of ALA is a measure of the high stability of the ME under environmental stress factors. Indeed, it can be seen in Figure 10 that generally the stability of the ME greatly increases regardless of the temperature (Figure 10a) and ionic strength (Figure 10b) after the introduction of CDs into the system indicating that a protective layer was practically formed by CDs, thus protecting the functional factor ALA in the ME. However, when the salinity was 4.5%, the loss ratio of ALA was higher than that calculated for a salinity of 0.9% in the CD−ALA-loaded ME. This result reveals that CDs in the water phase of the ME are sensitive to the ionic strength, and at a high concentration of salts, the interaction between the CDs and surfactant is weakened, leading the loss of ALA. An even more particular situation was noticed for pH 2 (Figure 10c), when the protective ability of the CDs was completely lost, so that the loss ratio of ALA in the CD−ALA-loaded ME was practically the same as that in the ALA-loaded ME. At this acidic pH value, both the amino and carboxyl groups of the CDs became protonated, hindering the interaction with the surfactant and thus destroying the ALA-protective layer. Effects of CDs on the In Vitro Release of ALA from CD−ALA-Loaded ME. As already demonstrated in the above results, the ALA molecule within ME could be well protected from oxygenation during the storage process. Subsequently, the main concern is that the ALA molecule could be released during digestion. Hence, we expected that the addition of CDs

Figure 10. Comparison of antioxidant stability between CD−ALAloaded ME and ALA-loaded ME under environmental stress factors: (a) different temperatures, (b) different salinities, and (c) different pHs.

to the oil−water interface could be turned-on. The in vitro release curves of ALA from ME at pH 2.0 and pH 7.0 are illustrated in Figure 11. It could be seen that although the released amounts of ALA increase with the increasing time regardless of the pH value, the release rate at pH 2.0 was about

Figure 11. In vitro release curves of ALA from CD−ALA−ME in PBS of pH 2.0 and pH 7.0. G

DOI: 10.1021/acs.jafc.8b01991 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry 4 times higher than that at pH 7.0, which was attributed to the fact that both amino and carboxyl groups on the CD surfaces were protonated in the acidic conditions of pH 2, leading to weakening of the interaction between the CDs and surfactant molecules; the protective layer formed by the CDs was thus turned-on, and ALA was rapidly released. However, at pH 7, ALA was gradually released because of the existence of the protective layer of CDs. This result was well in agreement with the analysis of antioxidant stability. From here, we see that CDs can protect ALA from oxidation during storage but do not prevent the release of ALA in vitro. Herein, we reported a novel oil-in-water ME system modified by carbon dots of 2−3 nm functionalized with −NH2 and −COOH groups on the surface. Regarding the potential use of the fabricated ME, the safety risk to the environment and human beings should be noticed. An and Liu reported that CDs may affect the function and character of DNA,35,36 whereas Yu reported that CDs have no toxicity to cells.37 In this work, CDs led the water phase in the modified ME to become tighter as the amount of CDs increased up to 2 mg/mL, and consequently, the particle sizes of the ME become smaller. Also, CDs are involved in interactions with the surfactant molecules at the oil−water interface. The formation of a clearly distinguished interface as a result of CD addition was proved by cryo-EM. The narrowing of the water layer and the tight interface substantially enhanced the antioxidant ability and anti-environmental-stress ability of the final ME containing the functional factor ALA. What is more valuable is that the CDs do not block the in vitro release of ALA from CD−ALA-loaded ME, and the release rate could be controlled by changing the pH of the buffer solution. The results have important implications for the development of effective delivery systems for bioactive components. The specialty of this delivery system is dependent on holding or releasing active ingredients properly by means of chemically manipulating the turn-off−turn-on oil−water interface during storage and release in the food industry. These findings might provide significant insight into developing efficient turn-on−turn-off switchable delivery systems for active functional factors. We are confident that the outcomes of this work will be extensively explored by researchers working particularly in functional food production to design new delivery systems for active ingredients.

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Xiaoxue Liu: 0000-0002-8234-9593 Chao Lu: 0000-0001-5364-2136 Sen Li: 0000-0002-2818-2286 Zhanzhong Wang: 0000-0002-9151-3308 Leping Dang: 0000-0003-1713-5422 Funding

We acknowledge financial support from the National Natural Science Foundation of China (21676196) and the Tianjin Municipal Natural Science Foundation (17JCYBJC20400). Notes

The authors declare no competing financial interest.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b01991. 1 H NMR spectra of ME, CD-loaded ME, ALA-loaded ME, and CD−ALA-loaded ME and colors of microemulsions loaded with different ALA concentrations (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86-022-27400291. Fax: +86-022-27400287. E-mail: [email protected] (Z.W.). *Tel.: +86-022-27400291. Fax: +86-022-27400287. E-mail: [email protected] (L.D.). ORCID

Mengna Hou: 0000-0002-8957-4582 Qing Li: 0000-0002-7678-4396 H

DOI: 10.1021/acs.jafc.8b01991 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.8b01991 J. Agric. Food Chem. XXXX, XXX, XXX−XXX