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The Effects of Surfactant and Metal Ions on the Stability and Rheological Properties of Nanoemulsions Loaded with Gardenia Yellow Pigment

The Effects of Surfactant and Metal Ions on the Stability and Rheological Properties of... Article The Effects of Surfactant and Metal Ions on the Stability and Rheological Properties of Nanoemulsions Loaded with Gardenia Yellow Pigment Li Gao and Bin Li * College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; gaoli19980711@hbeu.edu.cn * Correspondence: libinfood@mail.hzau.edu.cn; Tel.: +86-27-63730040; Fax: +86-27-87288636 † Current Institution: College of Life Science and Technology, Hubei Engineering University, Xiaogan 432000, China. Abstract: The present work reports the preparation of gardenia yellow pigment containing paraffin oil nanoemulsions stabilized by Span80 and Tween80. The preparation of the required nanoemulsions was optimized by testing different conditions, such as varying the hydrophilic–lipophilic balance (HLB), the emulsifier concentration (EC), the oil–water ratio (OWR), and the temperature (T), as determined by the average droplet diameter (ADD) and polydispersity index (PDI). Our results indicated that a minimum ADD of 65.9 nm and PDI of 0.116 were obtained at an optimum HLB value of 6.0, EC of 10% (w/w), OWR of 2:1, and T of 40 C. Both the steady-state and dynamic rheological parameters were further investigated, revealing that the emulsions exhibited pseudoplastic behaviors. The long-term stabilities of the nanoemulsions after the addition of inorganic salts were monitored by observing their visual appearances. It was found that the emulsions containing pure water or 0.1 M CaCl and AlCl became slightly separated, while the emulsions containing 0.1 M KCl and 2 3 NaCl showed no separation after 30 days of storage at room T. This difference among different salts could be related to the number of valence electrons of their cations. The spatial electrostatic effects of the monovalent cationic (KCl and NaCl) and the nonionic surfactants were greater than the delamination/sedimentation forces of the system, which was better than the salt based on the Citation: Gao, L.; Li, B. The Effects cations with valences greater than one (CaCl and AlCl ). In conclusion, the present work illustrated 2 3 of Surfactant and Metal Ions on the the formation, rheological properties, and stability of water containing gardenia yellow pigment in Stability and Rheological Properties paraffin oil nanoemulsions, which can be of great significance for the application of gardenia-yellow- of Nanoemulsions Loaded with pigment-based formulations. Gardenia Yellow Pigment. Appl. Nano 2023, 4, 61–74. https://doi.org/ Keywords: gardenia yellow pigment; nanoemulsion; hydrophilic–lipophilic balance; stability; rheology 10.3390/applnano4020005 Academic Editor: Angelo Maria Taglietti Received: 28 December 2022 1. Introduction Revised: 26 February 2023 Gardenia yellow pigment (GYP), extracted from Gardenia jasminoides Ellis, is a rare Accepted: 22 March 2023 natural water-soluble carotenoid consisting of crocin and crocetin [1]. Crocins are series Published: 4 April 2023 derivatives of crocetin glycosides [2] and are considered as the main components of water- soluble carotenoid in gardenia yellow, which is also used as a natural colorant. Moreover, it has been used as a natural food pigment for a long time, mainly for candy, noodles, jelly, and colored juice [3]. Crocetin, a type of carotenoid, contains a short carbon chain Copyright: © 2023 by the authors. and carboxyl groups at both ends of the carbon chain [4]. It is an aglycone of crocin Licensee MDPI, Basel, Switzerland. found in the fruit of gardenia [5] and is used as a traditional herbal medicine [4]. Crocetin This article is an open access article has many obvious effects in the treatment of diseases, such as cancer, sleep disturbances, distributed under the terms and and atherosclerosis [2]. Crocetin and crocins have received much attention in recent conditions of the Creative Commons years because of their health benefits and medical attributes, including their antioxidant Attribution (CC BY) license (https:// properties, anti-inflammatory properties, and their ability to reduce body fatigue and creativecommons.org/licenses/by/ 4.0/). Appl. Nano 2023, 4, 61–74. https://doi.org/10.3390/applnano4020005 https://www.mdpi.com/journal/applnano Appl. Nano 2023, 4 62 alleviate depression [5]. In light of the preceding discussion, gardenia yellow pigment-rich foods are regarded as beneficial for consumers’ health. Due to the coexistence of gardenoside, the gardenia yellow pigment easily fades and turns green when used as a food additive [6]. Gardenia yellow has been widely used as a natural pigment, but commercial gardenia yellow contains gardenoside, which may have negative effects [7]. Hence, it is crucial to prevent gardenia yellow from fading. A W/O nanoemulsion might be an effective way to overcome the stability-related limitations of gardenia yellow. Nanoemulsions are emulsions with droplet sizes in the range of 50–500 nm that are optically transparent and exhibit high kinetic stability [8]. The two main types of nanoemul- sions are oil-in-water (O/W) emulsions (which are oil phases dispersed in continuous aqueous phases), and water-in-oil (W/O) emulsions (which are water phases dispersed in continuous oil phases) [9]. These emulsions can be stabilized by the interfacial layer of the emulsifier and co-emulsifier [10–12]. The instability mechanisms of the nanoemul- sions in various breakdown processes are mainly Ostwald ripening, coalescence, and flocculation [13]. The emulsifier composition, droplet size, and surface charge are directly related to the stability of the nanoemulsion. Nanoemulsions are gaining increasing atten- tion due to their wide applications in the pharmaceutical, cosmetic, and food industries [14]. They have significant effects on the antioxidant activities of different bioactive compounds such as flavanols, resveratrol, and green tea polyphenols [15]. The W/O nanoemulsions’ combination of nonionic emulsifiers can encapsulate bioactive compounds to protect and increase their properties, including stability, solubility, and antioxidativity [15]. Liquid paraffin or mineral oil is a highly refined petroleum derivative composed of saturated hydrocarbons and is widely used as an oral laxative in Lesotho [16,17]. This oil protects against constipation and encopresis, which may be attributed to its tolerability and fewer side effects [18]. Studies have shown that it is good for the skin and development of babies, as well as for mothers with depression [19]. Moreover, it has been used in lip care cosmetics and skin for decades due to its wide range of viscosity options, high protection and cleaning properties, and excellent skin tolerance. Compared with vegetable oils, paraffin oils are nonallergenic because they are very stable and do not easily oxidize or become rancid. They have a long history of safe use, which has been confirmed by clinical and epidemiological geological data [20]. Therefore, a liquid-paraffin-based nanoemulsion can be better utilized in cosmetics and medical applications. In the present study, water containing gardenia yellow pigment in paraffin oil na- noemulsions was prepared and stabilized by a combination of Span80 and Tween80 surfac- tants. The main objective of the present study was to evaluate the stability of the emulsions formed at the optimum operating condition and to study the effects of the preparation parameters, e.g., the hydrophilic-lipophilic balance (HLB), the surfactant concentration, the oil-water ratio (OWR), the temperature (T), and the presence of inorganic salts on the stabil- ity of these emulsions. Furthermore, orthogonal and laser diffraction measurements were conducted to minimize the average droplet diameter (ADD) and polydispersity index (PDI) in order to select the most stable emulsion system for loading the hydrophilic gardenia yellow pigment. 2. Experimental Section 2.1. Materials Crocin standard (98% content) was purchased from the Institute for the Verification of Pharmaceutical and Biological Products (Beijing, China). Liquid paraffin, Span80 and Tween80 surfactants, anhydrous calcium chloride, sodium chloride, potassium chloride, and aluminum chloride were obtained from Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China) and were of analytical grade. Deionized water (Ultrapure, Thermo Fisher, Shanghai, China) was used in all the experiments. Appl. Nano 2023, 4 63 2.2. Methods 2.2.1. Preparation of the Emulsions The nanoemulsions were prepared using deionized water as the dispersed phase and paraffin oil as the continuous phase. First, the Tween80 and Span80 surfactants with different concentrations (4, 6, 8, 10, 12, 14, 16, 18, 20, and 30 wt%) at the required HLB values (4.5, 5, 5.5, 6, 6.5, and 7.0) were dispersed in liquid paraffin as the oil phase. The mixture was then heated to 50 C for 30 min in a water bath. After cooling to room T (20–25 C), the oil phase was stirred at 1000 rpm for 10 min (20 C) using magnetic stirring, and the aqueous phase was added to the oil phase dropwise to form an emulsion in a 250-mL conical flask that contained gardenia yellow pigment (1.6 g) and NaCl solution (0.8 g) with the concentrations of 0.1 mg/mL and 0.1 mol/L, respectively. The addition was carried out in such a way that all of the water phase was added at a rate of 2 drops/s. The magnetic stirring was continued for an additional time at 2000 rpm at 40 C. Once the stirring was complete, a white nanoemulsion was obtained. 2.2.2. Selection of HLB Values To obtain a stable emulsion system, Span80 (HLB: 4.3) and Tween80 (HLB: 15.0) were mixed at the desired ratio to obtain HLB values in the 4.5–7.0 range (e.g., 4.5, 5, 5.5, 6, 6.5, and 7.0). The required HLB values of the oil phases were calculated according to the Griffin method, expressed as follows [21]: W  H LB + W  H LB A A B B H LB = (1) AB W + W A B where HLB is the HLB of the mixed surfactants A and B; HLB and HLB are the HLB AB A B values of surfactants A and B, respectively; and W and W are the weight percentages of A B A and B, respectively. 2.2.3. Optimization of the Preparation Conditions In this study, W/O nanoemulsions were prepared by a low-energy emulsification method using the ADD and PDI as the evaluation criteria. Based on previous work [22], we first selected the HLB values (4.5, 5, 5.5, 6, 6.5, and 7.0), emulsifier concentrations (EC) (4, 6, 8, 10, 12, 14, 16, 18, 20, and 30 wt%), OWR (4:1, 3.5:1, 3:1, 2.5:1, 2:1, and 1:1 (w/w)), preparation T (30, 40, 50, 60, and 70 C), and stirring times (20, 30, 40, 50, and 60 min) to perform a single factor test. 2.2.4. Optimization of the Formulas Based on the examination of the single factors of the preparation conditions, we designed an L9 (34) orthogonal test to obtain the optimal formula composition by range analysis. 2.2.5. Determination of the Droplet Size and PDI The ADD and PDI were determined using a laser particle size analyzer (Malvern Instru- ments, Malvern, UK). The samples were diluted in paraffin oil 1000 times (10 L/10 mL) and stirred for 10 min at 1000 rpm using a vortex oscillator (Beijing United Keyi Technology Co., Ltd., MX-F, Beijing, China). After 12 h, the measurements were performed at 25 2 C and repeated in triplicate. The refractive index of the dispersed phase (deionized water) was 1.333 and that of the continuous phase (liquid paraffin) was 1.467. 2.2.6. Rheological Analysis Rheological measurements were conducted using an AR2000ex rheometer (TA In- struments, New Castle, PA, USA) with a parallel plate (diameter 40 mm, gap 1.0 mm). Continuous flow tests were carried out. The continuous ramp step was selected as the test type, and the shear rate ranged from 0 to 120 s within 2 min. Strain scanning mea- surements were performed in the range of 0.01 to 100% at the frequency of 1 rad/s to Appl. Nano 2023, 4 64 obtain the elastic (G ) and loss (G”) moduli and compare the rheological parameters of different emulsions. 2.2.7. Long-Term Stability Using the droplet size as the criterion, we evaluated the long-term stability of the W/O nanoemulsions prepared under the optimum conditions. The samples were sealed at room T for up to 30 days, and the particle sizes were measured at different time intervals. 2.2.8. Statistical Analysis All the experiments were performed three times, and the data were expressed as mean  SD (standard deviation). The single factor experimental data and statistical analy- sis were processed using Microsoft Excel 2007, and the orthogonal design and results were processed using the DPS v7.05 software. 3. Results and Discussion 3.1. Influence of the HLB Value The HLB value is a useful parameter for selecting a nonionic surfactant, which affects the size distribution and particle size formation of a stable nanoemulsion. The PDI was used to investigate the dispersibility of the nanoemulsion, with a smaller value indicating better dispersion. Typically, emulsifiers with lower HLB values (<7.0) are suitable for the fabrication of W/O emulsions, whereas emulsifiers with higher HLB values (>7.0) are suitable for the preparations of O/W emulsions [23]. To obtain the required HLB value, Span80 and Tween80 were mixed at the desired ratios to obtain HLB values in the range of 4.5–7.0 (4.5, 5.0, 5.5, 6.0, 6.5, and 7.0). As shown in Figure 1, when the HLB values increased from 4.5 to 6.0, the droplet size decreased from 129.33 to 89.24 nm. At lower HLB values, the mass fraction of Span80 was higher than that of Tween80, leading to the increased hydrophobicity of the mixed Appl. Nano 2023, 4, FOR PEER REVIEW 5 surfactant system, which could form a weak packing of the emulsifier molecules around the interface because of the imbalance between the hydrophobicity and hydrophilicity. Figure 1. Effects of HLB on the stability of the emulsions. Figure 1. Effects of HLB on the stability of the emulsions. However, due to the higher interfacial molecular area of Tween80 (Tween80: 2.48 nm ; However, due to the higher interfacial molecular area of Tween80 (Tween80: 2.48 Span80: 0.46 nm ), as the HLB values increased from 6.5 to 7.0, the droplet sizes did not 2 2 nm ; Span80: 0.46 nm ), as the HLB values increased from 6.5 to 7.0, the droplet sizes did significantly decrease. These results demonstrated that Tween80 and Span80 were in a not significantly decrease. These results demonstrated that Tween80 and Span80 were in saturated state on the droplet interface at an HLB of 6. At higher HLB values, excess a saturated state on the droplet interface at an HLB of 6. At higher HLB values, excess Tween80 molecules were present and occupied the largest active sites, which led to a lack of further reduction in the droplet size for HLB values greater than 6.0 [24,25]. The mini- mum particle size and PDI were found at the HLB values of 5.0 and 6.0, respectively. Therefore, the HLB values in the 5.0–6.0 range were selected for further experiments. 3.2. Influence of EC The EC was varied in the 4–30 wt% range at a fixed HLB of 5.0. According to the surface tension theory, emulsification occurs by reducing the interfacial tension between the oil and water phases. However, an examination of the data presented in Figure 2 shows that there were no significant differences between the results obtained for the ECs of 4 and 6 wt%. The nanoemulsion gradually became layered below 6 wt% because here the concentration of emulsifier was not sufficient to decrease the interfacial tension, which led to the coalescence of aqueous droplets. At the fraction of 16 wt%, the prepared emul- sion was translucent, whereas at a higher fraction (>16 wt%), the emulsion was more tur- bid. These results indicate that, after the critical micelle concentration was reached, the interfacial tension was not changed by the higher concentration of the surfactant [26]. Appl. Nano 2023, 4 65 Tween80 molecules were present and occupied the largest active sites, which led to a lack of further reduction in the droplet size for HLB values greater than 6.0 [24,25]. The minimum particle size and PDI were found at the HLB values of 5.0 and 6.0, respectively. Therefore, the HLB values in the 5.0–6.0 range were selected for further experiments. 3.2. Influence of EC The EC was varied in the 4–30 wt% range at a fixed HLB of 5.0. According to the surface tension theory, emulsification occurs by reducing the interfacial tension between the oil and water phases. However, an examination of the data presented in Figure 2 shows that there were no significant differences between the results obtained for the ECs of 4 and 6 wt%. The nanoemulsion gradually became layered below 6 wt% because here the concentration of emulsifier was not sufficient to decrease the interfacial tension, which led to the coalescence of aqueous droplets. At the fraction of 16 wt%, the prepared emulsion was Appl. Nano 2023, 4, FOR PEER REVIEW 6 translucent, whereas at a higher fraction (>16 wt%), the emulsion was more turbid. These results indicate that, after the critical micelle concentration was reached, the interfacial tension was not changed by the higher concentration of the surfactant [26]. Figure 2. Effect of EC on the stability of the emulsions. Figure 2. Effect of EC on the stability of the emulsions. Furthermore, an increase in the EC resulted in a decrease in the PDI. As the emulsifier fraction increased from 8 to 16 wt%, the PDI decreased from 0.283 to 0.017, demonstrating Furthermore, an increase in the EC resulted in a decrease in the PDI. As the emulsifier that the formulations prepared at low mass fractions were unstable [14]. fraction increased from 8 to 16 wt%, the PDI decreased from 0.283 to 0.017, demonstrating that the formulations prepared at low mass fractions were unstable [14]. 3.3. Influence of the Oil–Water Ratio (OWR) The oil–water mass ratio will affect the viscosity of the system and the stability of the 3.3. Influence of the Oil–Water Ratio (OWR) emulsion. The oil–water mass ratio was varied in the range from 1:1 to 4:1 at a fixed HLB The oil–water mass ratio will affect the viscosity of the system and the stability of the of 5 and surfactant concentration of 12% (w/w). As shown in Figure 3, it can be observed emulsion. The oil–water mass ratio was varied in the range from 1:1 to 4:1 at a fixed HLB that droplet size decreased from 154.0 nm to 124.0 nm on increasing ratios from 1:1 to of 5 and surfactant concentration of 12% (w/w). As shown in Figure 3, it can be observed 4:1 and the PDI exhibited no big difference. A higher ratio resulted in smaller droplet that droplet size decreased from 154.0 nm to 124.0 nm on increasing ratios from 1:1 to 4:1 size due to decrease in the viscosity of emulsion, and small viscosity is beneficial to the and the PDI exhibited no big difference. A higher ratio resulted in smaller droplet size dispersion of droplets [27]. The emulsions prepared at a ratio of 1:1 to 1.5:1 were found to be unstable after 24 h of storage at room temperature, whereas the emulsions prepared due to decrease in the viscosity of emulsion, and small viscosity is beneficial to the disper- at a ratio of 2:1 to 4:1 were more stable under the same conditions, respectively, and the sion of droplets [27]. The emulsions prepared at a ratio of 1:1 to 1.5:1 were found to be most stable emulsion appeared at the 3:1 oil–water mass ratio. It indicated that emulsions unstable after 24 h of storage at room temperature, whereas the emulsions prepared at a of a higher oil–water ratio reduced the viscosity of the system, leading to dispersing the ratio of 2:1 to 4:1 were more stable under the same conditions, respectively, and the most droplets sufficiently, which could make the droplet size smaller. stable emulsion appeared at the 3:1 oil–water mass ratio. It indicated that emulsions of a higher oil–water ratio reduced the viscosity of the system, leading to dispersing the drop- lets sufficiently, which could make the droplet size smaller. Figure 3. Effects of different OWRs on stability of the emulsions. Appl. Nano 2023, 4, FOR PEER REVIEW 6 Figure 2. Effect of EC on the stability of the emulsions. Furthermore, an increase in the EC resulted in a decrease in the PDI. As the emulsifier fraction increased from 8 to 16 wt%, the PDI decreased from 0.283 to 0.017, demonstrating that the formulations prepared at low mass fractions were unstable [14]. 3.3. Influence of the Oil–Water Ratio (OWR) The oil–water mass ratio will affect the viscosity of the system and the stability of the emulsion. The oil–water mass ratio was varied in the range from 1:1 to 4:1 at a fixed HLB of 5 and surfactant concentration of 12% (w/w). As shown in Figure 3, it can be observed that droplet size decreased from 154.0 nm to 124.0 nm on increasing ratios from 1:1 to 4:1 and the PDI exhibited no big difference. A higher ratio resulted in smaller droplet size due to decrease in the viscosity of emulsion, and small viscosity is beneficial to the disper- sion of droplets [27]. The emulsions prepared at a ratio of 1:1 to 1.5:1 were found to be unstable after 24 h of storage at room temperature, whereas the emulsions prepared at a ratio of 2:1 to 4:1 were more stable under the same conditions, respectively, and the most stable emulsion appeared at the 3:1 oil–water mass ratio. It indicated that emulsions of a Appl. Nano 2023, 4 66 higher oil–water ratio reduced the viscosity of the system, leading to dispersing the drop- lets sufficiently, which could make the droplet size smaller. Appl. Nano 2023, 4, FOR PEER REVIEW 7 Figure 3. Effects of different OWRs on stability of the emulsions. However, under a smaller oil and water ratio, the water content was relatively exces- Figure 3. Effects of different OWRs on stability of the emulsions. sive and additional water led to parts of emulsifiers in the system dispersed in it, which However, under a smaller oil and water ratio, the water content was relatively exces- resulted in insufficient emulsification of liquid paraffin wax, so the particle size of the sive and additional water led to parts of emulsifiers in the system dispersed in it, which emulsion became larger. resulted in insufficient emulsification of liquid paraffin wax, so the particle size of the emulsion became larger. 3.4. Influence of T 3.4. Influence To study ofthe T effects of T, the T was varied in the 30–70 °C range at a fixed HLB of 8.0, EC of 12 wt%, and OWR of 3:1. The T had a significant effect on the droplet size. As shown To study the effects of T, the T was varied in the 30–70 C range at a fixed HLB of in Figure 4, upon increasing the T from 30 to 70 °C, the droplet size increased from 119.36 8.0, EC of 12 wt%, and OWR of 3:1. The T had a significant effect on the droplet size. As to 189.08 nm, and the PDI also increased from 0.015 to 0.035. This indicated that a higher shown in Figure 4, upon increasing the T from 30 to 70 C, the droplet size increased from 119.36 to 189.08 nm, and the PDI also increased from 0.015 to 0.035. This indicated that T influenced the evaporation rate of the organic solvent [28], and the gradual dehydration a higher T influenced the evaporation rate of the organic solvent [28], and the gradual of emulsifier at the oil–water interface gave rise to the increase in interfacial tension, which dehydration of emulsifier at the oil–water interface gave rise to the increase in interfacial promoted the coalescence of liquid droplets [29]. tension, which promoted the coalescence of liquid droplets [29]. Figure 4. Effect of preparation T on stability of the emulsions. Figure 4. Effect of preparation T on stability of the emulsions. 3.5. Orthogonal Experiments Design 3.5. Orthogonal Experiments Design Various methods, such as the single factor method and the response surface method [30], Various methods, such as the single factor method and the response surface method were used to optimize the experimental conditions. In the present study, to reduce the [30], were used to optimize the experimental conditions. In the present study, to reduce experimental time, orthogonal experiments, which can be used to assess the importance the experimental time, orthogonal experiments, which can be used to assess the im- portance of each factor and determine the ideal values, were performed for the optimiza- tion of the preparation conditions of the W/O emulsions [31,32]. In the preparation process of the W/O emulsions, the preparation conditions were optimized by L9 (34) orthogonal experiments using the ADD and the PDI as the assessment indices. The HLB value (A), EC (B), OWR (C), and preparation T (PT (D)) were selected as the experimental factors. Each factor was designed with three levels, and the factors and levels of the orthogonal tests are listed in Table 1, with the results shown in Table 2. Table 1. Factors and levels of Orthogonal experiment. Factor Level HLB EC (wt%) OWR (w/w) T (°C) A B C D 1 5.0 10 2:1 40 2 5.5 12 3:1 50 3 6.0 16 4:1 60 Appl. Nano 2023, 4 67 of each factor and determine the ideal values, were performed for the optimization of the preparation conditions of the W/O emulsions [31,32]. In the preparation process of the W/O emulsions, the preparation conditions were optimized by L9 (34) orthogonal experiments using the ADD and the PDI as the assessment indices. The HLB value (A), EC (B), OWR (C), and preparation T (PT (D)) were selected as the experimental factors. Each factor was designed with three levels, and the factors and levels of the orthogonal tests are listed in Table 1, with the results shown in Table 2. Table 1. Factors and levels of Orthogonal experiment. Factor Level HLB EC (wt%) OWR (w/w) T ( C) A B C D 1 5.0 10 2:1 40 2 5.5 12 3:1 50 3 6.0 16 4:1 60 Table 2. Results of the Orthogonal experiment. HLB EC (wt%) OWR (w/w) T ( C) Run ADD (nm) PDI A B C D 1 1 1 1 1 120.432 0.032 2 1 2 2 2 141.105 0.172 3 1 3 3 3 189.306 0.148 4 2 1 2 3 130.423 0.152 5 2 2 3 1 117.406 0.082 6 2 3 1 2 141.323 0.011 7 3 1 3 2 99.207 0.225 8 3 2 1 3 94.104 0.573 9 3 3 2 1 104.413 0.204 k 150.28 116.69 118.62 114.08 k 129.72 117.54 125.31 127.21 ADD k 99.24 145.01 135.31 137.94 R 51.04 28.33 16.69 23.86 order of importance A > B > D > C optimal level A B D C 3 1 1 1 k 0.12 0.14 0.21 0.11 k 0.08 0.28 0.14 0.14 PDI k 0.33 0.12 0.15 0.29 R 0.25 0.16 0.06 0.19 order of importance A > D > B > C optimal level A D B C 2 1 3 2 As shown in Table 2, the PDI values of runs 7, 8, and 9 were 0.225, 0.573, and 0.204, respectively, while those of the other runs were less than 0.2. The ADD values of runs 7 and 8 were 99.207 and 94.104 nm, respectively, while those of the other runs exceeded 100 nm. The results of the range analysis used to determine the optimal levels of the factors are shown in Table 2. The K/k value for each level was the average of three ADD values and the polydispersity index (PDI), as shown in Table 2. By comparing different K/k values, the optimal level of factors for the preparation of the W/O nanoemulsions could be identified. In this work, the minimum K/k value was better for the optimal level. The difference between the maximum and minimum K/k values is represented by the range value (R), which reflects the detrimental effects of the level on the ADD and PDI. The most important factor was obtained when R was maximal, and the order of im- portance was A > B > D > C and A > D > B > C for the ADD and PDI, respectively [33,34]. Appl. Nano 2023, 4 68 Considering the practicability of the W/O emulsions, the ADD was the most important factor for the formulation selection. The factors influencing the ADD were in the order of HLB value > EC > emulsification T > OWR. The final optimal preparation factors were a HLB of 6.0, EC of 10 wt%, OWR of 2:1, and emulsification T of 40 C. However, the optimal combination was not listed in the nine runs (Table 2), and three additional validation experiments showed that the ADD and the PDI were 45.15 nm and 0.25, respectively, under the optimal preparation conditions (Table 3). These values were smaller than those obtained in any of the nine runs, which confirmed the reliability of the model prediction by the array. Table 3. Confirmatory experiment. Factor HLB EC (wt%) OWR (w/w) T ( C) condition 6 10 2:1 40 times once second third mean Appl. Nano 2023, 4, FOR PEER REVIEW 9 ADD (nm) 73.17 65.91 65.83 45.15 PDI 0.27 0.12 0.3 0.25 Table 3. Confirmatory experiment. Notably, as shown in Figure 5, the laser diffraction measurements exhibited that the W/O nanoemulsion particle size distribution was relatively uniform, and the width was Factor HLB EC (wt%) OWR (w/w) T (°C) substantially unchanged. This indicated that the adjustment of the proportions of the added condition 6 10 2:1 40 materials could significantly reduce the interfacial tension of the water and oil, such that times once second third mean the Ostwald ripening rate was weak, physical changes such as Brownian motion could be ADD (nm) 73.17 65.91 65.83 45.15 resisted, and the nanoemulsion stability could be improved. The more stable the emulsion PDI 0.27 0.12 0.3 0.25 system, the more obvious the protective effect of the gardenia yellow pigment. Figure 5. Particle size distribution of nanoemulsion with the confirmatory experiment. Figure 5. Particle size distribution of nanoemulsion with the confirmatory experiment. 3.6. Rheology 3.6. Rheology 3.6.1. Shear Flow Tests 3.6.1. Shear Flow Tests Research has revealed that high emulsion viscosity is conducive to its stability [35]. As Research has revealed that high emulsion viscosity is conducive to its stability [35]. shown in Figure 6a, the viscosity decreased significantly with the increase in the shear rate. As shown in Figure 6a, the viscosity decreased significantly with the increase in the shear At higher shear rates, the viscosity tended to be almost constant, indicating that the W/O rate. At higher shear rates, the viscosity tended to be almost constant, indicating that the nanoemulsion was a pseudoplastic fluid with shear thinning behavior [36]. This was due W/O nanoemulsion was a pseudoplastic fluid with shear thinning behavior [36]. This was to the higher shear strain leading to more obvious elongation, deformation, and network due to the higher shear strain leading to more obvious elongation, deformation, and net- fracture of the nanoemulsion droplets [37]. work fracture of the nanoemulsion droplets [37]. (a) Appl. Nano 2023, 4, FOR PEER REVIEW 9 Table 3. Confirmatory experiment. Factor HLB EC (wt%) OWR (w/w) T (°C) condition 6 10 2:1 40 times once second third mean ADD (nm) 73.17 65.91 65.83 45.15 PDI 0.27 0.12 0.3 0.25 Figure 5. Particle size distribution of nanoemulsion with the confirmatory experiment. 3.6. Rheology 3.6.1. Shear Flow Tests Research has revealed that high emulsion viscosity is conducive to its stability [35]. As shown in Figure 6a, the viscosity decreased significantly with the increase in the shear rate. At higher shear rates, the viscosity tended to be almost constant, indicating that the W/O nanoemulsion was a pseudoplastic fluid with shear thinning behavior [36]. This was due to the higher shear strain leading to more obvious elongation, deformation, and net- Appl. Nano 2023, 4 69 work fracture of the nanoemulsion droplets [37]. Appl. Nano 2023, 4, FOR PEER REVIEW 10 (a) (b) Figure 6. The relationship between viscosity and shear rate for optimal nanoemulsion (a); The Figure 6. The relationship between viscosity and shear rate for optimal nanoemulsion (a); The change change of elastic modulus and viscous modulus with frequency (b). of elastic modulus and viscous modulus with frequency (b). 3.6.2. Oscillation Strain Sweep 3.6.2. Oscillation Strain Sweep Oscillation strain sweep tests were conducted to characterize the structural strengths of Oscillation strain sweep tests were conducted to characterize the structural strengths of the emulsions in the linear viscoelastic region. Figure 6b shows the frequency dependence the emulsions in the linear viscoelastic region. Figure 6b shows the frequency dependence of the dynamic modulus. The storage modulus (G′) is a measure of the energy stored elas- of the dynamic modulus. The storage modulus (G ) is a measure of the energy stored tically during an oscillation cycle, whereas the loss modulus (G″) is a measure of the energy elastically during an oscillation cycle, whereas the loss modulus (G”) is a measure of the dissipated by the viscous flow during the oscillation. The G′ modulus was negligible com- energy dissipated by the viscous flow during the oscillation. The G modulus was negligible pacompar red to the ed G to″the for t G” he for nanoemu the nanoemulsions lsions with 1 with 0% 10% (w/w()w s/ uw rf)ac surfactant tant concentr concentrations; ations; thus, thus, the emulsions exhibited a predominant viscous behavior. the emulsions exhibited a predominant viscous behavior. However However, , as as can can be be s seen een fr from om Figur Figure e 7 7, , when when th the e surfactant surfactant concentration w concentration was as 20% 20% and and 2 24% 4% ( (w w/ /w w), ), the the G G′ v value alue o of f the the two two em emulsions ulsions w was as ab about out twic twice e as as high high as as t that hat o of f G G”,″, indicating that the emulsion had a certain elasticity. The modulus of elasticity depended on indicating that the emulsion had a certain elasticity. The modulus of elasticity depended the types of interaction forces between the droplets and their nearest neighbors—attractive on the types of interaction forces between the droplets and their nearest neighbors—at- or trar cepulsive tive or rep [38 uls ,39 iv]. e [ The 38,39 change ]. The change from a pr from edominantl a predom y iviscous nantly vto iscou a pr s to edominant a predom lyinan elastic tly elastic response was affected by the ratio of the adsorption thickness of the surfactant to the droplet radius R [39]. Appl. Nano 2023, 4 70 Appl. Nano 2023, 4, FOR PEER REVIEW 11 response was affected by the ratio of the adsorption thickness of the surfactant to the droplet radius R [39]. (a) (b) Figure 7. dynamic (a,b) rheological analyses of W/O emulsions with different ECs. (a) dynamic rhe- Figure 7. Dynamic (a,b) rheological analyses of W/O emulsions with different ECs. (a) dynamic ological analysis of W/O emulsions with different ECs and elastic moduli G′, and (b) dynamic rhe- rheological analysis of W/O emulsions with different ECs and elastic moduli G , and (b) dynamic ological analysis of W/O emulsions with different ECs and loss moduli G″. rheological analysis of W/O emulsions with different ECs and loss moduli G”. 3.7. Stability Evaluation of the Emulsions 3.7. Stability Evaluation of the Emulsions In this study, the effects of different chloride salts (sodium, potassium, aluminum, In this study, the effects of different chloride salts (sodium, potassium, aluminum, and and calcium) at 0.1 M concentrations on the long-term stability of the W/O nanoemulsions calcium) at 0.1 M concentrations on the long-term stability of the W/O nanoemulsions were were examined. As shown in Figure 8, the addition of chloride salts in the aqueous phase examined. As shown in Figure 8, the addition of chloride salts in the aqueous phase had had little effect on the initial ADD and did not result in significant differences in the sta- little effect on the initial ADD and did not result in significant differences in the stability bility compared to the systems with no added salt. This indicated that the EC was the compared to the systems with no added salt. This indicated that the EC was the main main determinant of the initial particle size of the W/O nanoemulsions. As shown in Fig- determinant of the initial particle size of the W/O nanoemulsions. As shown in Figure 9, ure 9, after 30 days of observation at room T, the emulsions containing pure water or 0.1 after 30 days of observation at room T, the emulsions containing pure water or 0.1 M CaCl M CaCl2 and AlCl3 slightly separated, whereas the emulsions containing 0.1 M KCl and and AlCl slightly separated, whereas the emulsions containing 0.1 M KCl and NaCl did NaCl did not show any separation until 30 days at room T. This difference was related to the number of valence electrons of the cations of these salts. Appl. Nano 2023, 4 71 Appl. Nano 2023, 4, FOR PEER REVIEW 12 Appl. Nano 2023, 4, FOR PEER REVIEW 12 not show any separation until 30 days at room T. This difference was related to the number of valence electrons of the cations of these salts. Figure 8. Long-term stability of pure water nanoemulsions and nanoemulsions with 0.1 M salts. Figure 8. Long-term stability of pure water nanoemulsions and nanoemulsions with 0.1 M salts. Figure 8. Long-term stability of pure water nanoemulsions and nanoemulsions with 0.1 M salts. Figure 9. Appearance of pure water nanoemulsions and nanoemulsions with 0.1 M salts after stor- Figure 9. Appearance of pure water nanoemulsions and nanoemulsions with 0.1 M salts after stor- Figure 9. Appearance of pure water nanoemulsions and nanoemulsions with 0.1 M salts after storage age for 30 days. The type of salt from left to right are pure water, AlCl3, CaCl2, NaCl, and KCl. age for 30 days. The type of salt from left to right are pure water, AlCl3, CaCl2, NaCl, and KCl. for 30 days. The type of salt from left to right are pure water, AlCl , CaCl , NaCl, and KCl. 3 2 KCl and NaCl are ionic compounds, and upon addition to the water phase, these KCl KCl and and NaCl NaCl are are ionic ionic compounds, compounds, a and nd upon upon add addition ition to to the the wate waterr phase, phase, these these + + + + − + + − crystals dissociate into Na , K , and Cl ions in water. It is well known that the emulsifica- crystals dissociate into Na , K , and Cl ions in water. It is well known that the emul- crystals dissociate into Na , K , and Cl ions in water. It is well known that the emulsifica- tion process includes the break-up of droplets and the re-coalescence of the newly formed sification process includes the break-up of droplets and the re-coalescence of the newly tion process includes the break-up of droplets and the re-coalescence of the newly formed droplets. The investigations by Jafari et al. [40]. showed that the reduction in the re-coa- formed droplets. The droplets. invest The ig inv ation estigations s by Jafari e by tJafari al. [40]. show et al. [40]. ed th showed at the re that duction in the reduction the re-co in the a- lescence w re-coalescence as strong was str er th onger an the enhanc than the enhancement ement of of the the drop droplet let ddisr isrup uption. tion. Acco Accor rding ding to to lescence was stronger than the enhancement of the droplet disruption. According to T Ta adr dros os [ [41 41], ], t the he s stability tability of of the the oi oil–water l–water in interface terface dep depends ends on on the the e electr lectros ostatic tatic or or sp spatial atial Tadros [41], the stability of the oil–water interface depends on the electrostatic or spatial repul repulsion sion in interact teractions ions. . The Ther refore, efore, when when KCl KCl and and N NaCl aCl d dissociated issociated iinto nto ions ions in in w water ater,, due due repulsion interactions. Therefore, when KCl and NaCl dissociated into ions in water, due to to the the re reduced duced re re-coalescence, -coalescence, the these se ion ions s g gave ave r rise ise to to repul repulsive sive in interactions teractions in in th the e drop droplets lets to the reduced re-coalescence, these ions gave rise to repulsive interactions in the droplets that could improve the stability of the oil–water interface. This conclusion agrees with the that could improve the stability of the oil–water interface. This conclusion agrees with the that could improve the stability of the oil–water interface. This conclusion agrees with the results reported by Scherze et al. [42]. results reported by Scherze et al. [42]. results reported by Scherze et al. [42]. For the effect of CaCl and AlCl on the nanoemulsion stability, it was suggested For the effect of CaCl2 and 2 AlCl3 on the nanoemu 3 lsion stability, it was suggested that For the effect of CaCl2 and AlCl3 on the nanoemulsion stability, it was suggested that that a reduction in the attractive force between the water droplets due to the addition of a reduction in the attractive force between the water droplets due to the addition of the a reduction in the attractive force between the water droplets due to the addition of the the electrolytes in the aqueous phase increased the W/O emulsion stability with respect electrolytes in the aqueous phase increased the W/O emulsion stability with respect to the electrolytes in the aqueous phase increased the W/O emulsion stability with respect to the to the coalescence. Consequently, the attractive forces were minimized by matching the coalescence. Consequently, the attractive forces were minimized by matching the refrac- coalescence. Consequently, the attractive forces were minimized by matching the refrac- refractive indices and/or the dielectric constants of the two phases. As the electrolyte tive indices and/or the dielectric constants of the two phases. As the electrolyte concentra- tive indices and/or the dielectric constants of the two phases. As the electrolyte concentra- concentration increased, the refractive index of the aqueous phase increased, and the tion increased, the refractive index of the aqueous phase increased, and the ionization tion increased, the refractive index of the aqueous phase increased, and the ionization ionization constant decreased [43]. Thus, the addition of calcium and aluminum salts constant decreased [43]. Thus, the addition of calcium and aluminum salts decreased the constant decreased [43]. Thus, the addition of calcium and aluminum salts decreased the decreased the attractive forces, leading to the reduced frequency of collisions between attractive forces, leading to the reduced frequency of collisions between the water droplets, attractive forces, leading to the reduced frequency of collisions between the water droplets, the water droplets, improving the stability with respect to the coalescence of the W/O improving the stability with respect to the coalescence of the W/O emulsions, and leading improving the stability with respect to the coalescence of the W/O emulsions, and leading emulsions, and leading to their sedimentation. to their sedimentation. to their sedimentation. Appl. Nano 2023, 4 72 The above results demonstrated that the spatial electrostatic effects of the monovalent cationic (KCl and NaCl) and the nonionic surfactants were greater than the delamina- tion/sedimentation forces of the system, providing effective resistance to the destabilizing processes, such as Ostwald ripening, coalescence, and flocculation. Thus, the emulsions with NaCl and KCl had small particle sizes, narrow particle size distributions, and better long-term stability, whereas the nanoemulsions with salts based on cations with valences greater than one (CaCl and AlCl ) showed the opposite effects. 2 3 4. Conclusions In this work, gardenia-yellow-pigment-based W/O nanoemulsions were produced, and the effect of the surfactant and metal ions on the nanoemulsions was studied. The optimal W/O nanoemulsions’ preparation parameters were defined by single factor experi- ments and orthogonal tests, including the hydrophilic–lipophilic balance (HLB) value (6), the emulsifier concentration (10%), the oil–water ratio (2:1), and the preparation tempera- ture (40 C). The experimental analysis showed that the W/O nanoemulsions were shear thinning pseudoplastic fluids, and the rheology of the W/O nanoemulsions changed from viscosity to elasticity by adding the appropriate concentration of surfactant. In particular, the stability of the nanoemulsion was improved by electrostatic and spatial exclusion after NaCl and KCl addition, whereas the instability mechanism after CaCl and AlCl addition 2 3 resulted in coalescence and sedimentation. This preliminary study provides new insight into the design of new generation nanoemulsions containing gardenia yellow pigment for health promotion. The outcomes can be considered as the beginning, requiring further research focused on gardenia yellow pigment for commercial usage. Author Contributions: Conceptualization, L.G. and B.L.; methodology, L.G.; software, L.G.; valida- tion, L.G. and B.L.; formal analysis, L.G.; investigation, L.G.; resources, L.G. and B.L.; data curation, L.G.; writing—original draft preparation, L.G.; writing—review and editing, L.G. and B.L.; visualiza- tion, L.G.; supervision, L.G.; project administration, L.G.; funding acquisition, B.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was financially supported by the Hubei Provincial Natural Science Foundation major project (2009CDA102) from Hubei Province, China and the APC was funded by [2009CDA102]. Data Availability Statement: The data presented in this study are available from the authors. Acknowledgments: Li Gao and Bin Li contributed equally to this work. 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The Effects of Surfactant and Metal Ions on the Stability and Rheological Properties of Nanoemulsions Loaded with Gardenia Yellow Pigment

Gao, Li; Li, Bin
Applied Nano , Volume 4 (2) – Apr 4, 2023
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Abstract

Article The Effects of Surfactant and Metal Ions on the Stability and Rheological Properties of Nanoemulsions Loaded with Gardenia Yellow Pigment Li Gao and Bin Li * College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; gaoli19980711@hbeu.edu.cn * Correspondence: libinfood@mail.hzau.edu.cn; Tel.: +86-27-63730040; Fax: +86-27-87288636 † Current Institution: College of Life Science and Technology, Hubei Engineering University, Xiaogan 432000, China. Abstract: The present work reports the preparation of gardenia yellow pigment containing paraffin oil nanoemulsions stabilized by Span80 and Tween80. The preparation of the required nanoemulsions was optimized by testing different conditions, such as varying the hydrophilic–lipophilic balance (HLB), the emulsifier concentration (EC), the oil–water ratio (OWR), and the temperature (T), as determined by the average droplet diameter (ADD) and polydispersity index (PDI). Our results indicated that a minimum ADD of 65.9 nm and PDI of 0.116 were obtained at an optimum HLB value of 6.0, EC of 10% (w/w), OWR of 2:1, and T of 40 C. Both the steady-state and dynamic rheological parameters were further investigated, revealing that the emulsions exhibited pseudoplastic behaviors. The long-term stabilities of the nanoemulsions after the addition of inorganic salts were monitored by observing their visual appearances. It was found that the emulsions containing pure water or 0.1 M CaCl and AlCl became slightly separated, while the emulsions containing 0.1 M KCl and 2 3 NaCl showed no separation after 30 days of storage at room T. This difference among different salts could be related to the number of valence electrons of their cations. The spatial electrostatic effects of the monovalent cationic (KCl and NaCl) and the nonionic surfactants were greater than the delamination/sedimentation forces of the system, which was better than the salt based on the Citation: Gao, L.; Li, B. The Effects cations with valences greater than one (CaCl and AlCl ). In conclusion, the present work illustrated 2 3 of Surfactant and Metal Ions on the the formation, rheological properties, and stability of water containing gardenia yellow pigment in Stability and Rheological Properties paraffin oil nanoemulsions, which can be of great significance for the application of gardenia-yellow- of Nanoemulsions Loaded with pigment-based formulations. Gardenia Yellow Pigment. Appl. Nano 2023, 4, 61–74. https://doi.org/ Keywords: gardenia yellow pigment; nanoemulsion; hydrophilic–lipophilic balance; stability; rheology 10.3390/applnano4020005 Academic Editor: Angelo Maria Taglietti Received: 28 December 2022 1. Introduction Revised: 26 February 2023 Gardenia yellow pigment (GYP), extracted from Gardenia jasminoides Ellis, is a rare Accepted: 22 March 2023 natural water-soluble carotenoid consisting of crocin and crocetin [1]. Crocins are series Published: 4 April 2023 derivatives of crocetin glycosides [2] and are considered as the main components of water- soluble carotenoid in gardenia yellow, which is also used as a natural colorant. Moreover, it has been used as a natural food pigment for a long time, mainly for candy, noodles, jelly, and colored juice [3]. Crocetin, a type of carotenoid, contains a short carbon chain Copyright: © 2023 by the authors. and carboxyl groups at both ends of the carbon chain [4]. It is an aglycone of crocin Licensee MDPI, Basel, Switzerland. found in the fruit of gardenia [5] and is used as a traditional herbal medicine [4]. Crocetin This article is an open access article has many obvious effects in the treatment of diseases, such as cancer, sleep disturbances, distributed under the terms and and atherosclerosis [2]. Crocetin and crocins have received much attention in recent conditions of the Creative Commons years because of their health benefits and medical attributes, including their antioxidant Attribution (CC BY) license (https:// properties, anti-inflammatory properties, and their ability to reduce body fatigue and creativecommons.org/licenses/by/ 4.0/). Appl. Nano 2023, 4, 61–74. https://doi.org/10.3390/applnano4020005 https://www.mdpi.com/journal/applnano Appl. Nano 2023, 4 62 alleviate depression [5]. In light of the preceding discussion, gardenia yellow pigment-rich foods are regarded as beneficial for consumers’ health. Due to the coexistence of gardenoside, the gardenia yellow pigment easily fades and turns green when used as a food additive [6]. Gardenia yellow has been widely used as a natural pigment, but commercial gardenia yellow contains gardenoside, which may have negative effects [7]. Hence, it is crucial to prevent gardenia yellow from fading. A W/O nanoemulsion might be an effective way to overcome the stability-related limitations of gardenia yellow. Nanoemulsions are emulsions with droplet sizes in the range of 50–500 nm that are optically transparent and exhibit high kinetic stability [8]. The two main types of nanoemul- sions are oil-in-water (O/W) emulsions (which are oil phases dispersed in continuous aqueous phases), and water-in-oil (W/O) emulsions (which are water phases dispersed in continuous oil phases) [9]. These emulsions can be stabilized by the interfacial layer of the emulsifier and co-emulsifier [10–12]. The instability mechanisms of the nanoemul- sions in various breakdown processes are mainly Ostwald ripening, coalescence, and flocculation [13]. The emulsifier composition, droplet size, and surface charge are directly related to the stability of the nanoemulsion. Nanoemulsions are gaining increasing atten- tion due to their wide applications in the pharmaceutical, cosmetic, and food industries [14]. They have significant effects on the antioxidant activities of different bioactive compounds such as flavanols, resveratrol, and green tea polyphenols [15]. The W/O nanoemulsions’ combination of nonionic emulsifiers can encapsulate bioactive compounds to protect and increase their properties, including stability, solubility, and antioxidativity [15]. Liquid paraffin or mineral oil is a highly refined petroleum derivative composed of saturated hydrocarbons and is widely used as an oral laxative in Lesotho [16,17]. This oil protects against constipation and encopresis, which may be attributed to its tolerability and fewer side effects [18]. Studies have shown that it is good for the skin and development of babies, as well as for mothers with depression [19]. Moreover, it has been used in lip care cosmetics and skin for decades due to its wide range of viscosity options, high protection and cleaning properties, and excellent skin tolerance. Compared with vegetable oils, paraffin oils are nonallergenic because they are very stable and do not easily oxidize or become rancid. They have a long history of safe use, which has been confirmed by clinical and epidemiological geological data [20]. Therefore, a liquid-paraffin-based nanoemulsion can be better utilized in cosmetics and medical applications. In the present study, water containing gardenia yellow pigment in paraffin oil na- noemulsions was prepared and stabilized by a combination of Span80 and Tween80 surfac- tants. The main objective of the present study was to evaluate the stability of the emulsions formed at the optimum operating condition and to study the effects of the preparation parameters, e.g., the hydrophilic-lipophilic balance (HLB), the surfactant concentration, the oil-water ratio (OWR), the temperature (T), and the presence of inorganic salts on the stabil- ity of these emulsions. Furthermore, orthogonal and laser diffraction measurements were conducted to minimize the average droplet diameter (ADD) and polydispersity index (PDI) in order to select the most stable emulsion system for loading the hydrophilic gardenia yellow pigment. 2. Experimental Section 2.1. Materials Crocin standard (98% content) was purchased from the Institute for the Verification of Pharmaceutical and Biological Products (Beijing, China). Liquid paraffin, Span80 and Tween80 surfactants, anhydrous calcium chloride, sodium chloride, potassium chloride, and aluminum chloride were obtained from Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China) and were of analytical grade. Deionized water (Ultrapure, Thermo Fisher, Shanghai, China) was used in all the experiments. Appl. Nano 2023, 4 63 2.2. Methods 2.2.1. Preparation of the Emulsions The nanoemulsions were prepared using deionized water as the dispersed phase and paraffin oil as the continuous phase. First, the Tween80 and Span80 surfactants with different concentrations (4, 6, 8, 10, 12, 14, 16, 18, 20, and 30 wt%) at the required HLB values (4.5, 5, 5.5, 6, 6.5, and 7.0) were dispersed in liquid paraffin as the oil phase. The mixture was then heated to 50 C for 30 min in a water bath. After cooling to room T (20–25 C), the oil phase was stirred at 1000 rpm for 10 min (20 C) using magnetic stirring, and the aqueous phase was added to the oil phase dropwise to form an emulsion in a 250-mL conical flask that contained gardenia yellow pigment (1.6 g) and NaCl solution (0.8 g) with the concentrations of 0.1 mg/mL and 0.1 mol/L, respectively. The addition was carried out in such a way that all of the water phase was added at a rate of 2 drops/s. The magnetic stirring was continued for an additional time at 2000 rpm at 40 C. Once the stirring was complete, a white nanoemulsion was obtained. 2.2.2. Selection of HLB Values To obtain a stable emulsion system, Span80 (HLB: 4.3) and Tween80 (HLB: 15.0) were mixed at the desired ratio to obtain HLB values in the 4.5–7.0 range (e.g., 4.5, 5, 5.5, 6, 6.5, and 7.0). The required HLB values of the oil phases were calculated according to the Griffin method, expressed as follows [21]: W  H LB + W  H LB A A B B H LB = (1) AB W + W A B where HLB is the HLB of the mixed surfactants A and B; HLB and HLB are the HLB AB A B values of surfactants A and B, respectively; and W and W are the weight percentages of A B A and B, respectively. 2.2.3. Optimization of the Preparation Conditions In this study, W/O nanoemulsions were prepared by a low-energy emulsification method using the ADD and PDI as the evaluation criteria. Based on previous work [22], we first selected the HLB values (4.5, 5, 5.5, 6, 6.5, and 7.0), emulsifier concentrations (EC) (4, 6, 8, 10, 12, 14, 16, 18, 20, and 30 wt%), OWR (4:1, 3.5:1, 3:1, 2.5:1, 2:1, and 1:1 (w/w)), preparation T (30, 40, 50, 60, and 70 C), and stirring times (20, 30, 40, 50, and 60 min) to perform a single factor test. 2.2.4. Optimization of the Formulas Based on the examination of the single factors of the preparation conditions, we designed an L9 (34) orthogonal test to obtain the optimal formula composition by range analysis. 2.2.5. Determination of the Droplet Size and PDI The ADD and PDI were determined using a laser particle size analyzer (Malvern Instru- ments, Malvern, UK). The samples were diluted in paraffin oil 1000 times (10 L/10 mL) and stirred for 10 min at 1000 rpm using a vortex oscillator (Beijing United Keyi Technology Co., Ltd., MX-F, Beijing, China). After 12 h, the measurements were performed at 25 2 C and repeated in triplicate. The refractive index of the dispersed phase (deionized water) was 1.333 and that of the continuous phase (liquid paraffin) was 1.467. 2.2.6. Rheological Analysis Rheological measurements were conducted using an AR2000ex rheometer (TA In- struments, New Castle, PA, USA) with a parallel plate (diameter 40 mm, gap 1.0 mm). Continuous flow tests were carried out. The continuous ramp step was selected as the test type, and the shear rate ranged from 0 to 120 s within 2 min. Strain scanning mea- surements were performed in the range of 0.01 to 100% at the frequency of 1 rad/s to Appl. Nano 2023, 4 64 obtain the elastic (G ) and loss (G”) moduli and compare the rheological parameters of different emulsions. 2.2.7. Long-Term Stability Using the droplet size as the criterion, we evaluated the long-term stability of the W/O nanoemulsions prepared under the optimum conditions. The samples were sealed at room T for up to 30 days, and the particle sizes were measured at different time intervals. 2.2.8. Statistical Analysis All the experiments were performed three times, and the data were expressed as mean  SD (standard deviation). The single factor experimental data and statistical analy- sis were processed using Microsoft Excel 2007, and the orthogonal design and results were processed using the DPS v7.05 software. 3. Results and Discussion 3.1. Influence of the HLB Value The HLB value is a useful parameter for selecting a nonionic surfactant, which affects the size distribution and particle size formation of a stable nanoemulsion. The PDI was used to investigate the dispersibility of the nanoemulsion, with a smaller value indicating better dispersion. Typically, emulsifiers with lower HLB values (<7.0) are suitable for the fabrication of W/O emulsions, whereas emulsifiers with higher HLB values (>7.0) are suitable for the preparations of O/W emulsions [23]. To obtain the required HLB value, Span80 and Tween80 were mixed at the desired ratios to obtain HLB values in the range of 4.5–7.0 (4.5, 5.0, 5.5, 6.0, 6.5, and 7.0). As shown in Figure 1, when the HLB values increased from 4.5 to 6.0, the droplet size decreased from 129.33 to 89.24 nm. At lower HLB values, the mass fraction of Span80 was higher than that of Tween80, leading to the increased hydrophobicity of the mixed Appl. Nano 2023, 4, FOR PEER REVIEW 5 surfactant system, which could form a weak packing of the emulsifier molecules around the interface because of the imbalance between the hydrophobicity and hydrophilicity. Figure 1. Effects of HLB on the stability of the emulsions. Figure 1. Effects of HLB on the stability of the emulsions. However, due to the higher interfacial molecular area of Tween80 (Tween80: 2.48 nm ; However, due to the higher interfacial molecular area of Tween80 (Tween80: 2.48 Span80: 0.46 nm ), as the HLB values increased from 6.5 to 7.0, the droplet sizes did not 2 2 nm ; Span80: 0.46 nm ), as the HLB values increased from 6.5 to 7.0, the droplet sizes did significantly decrease. These results demonstrated that Tween80 and Span80 were in a not significantly decrease. These results demonstrated that Tween80 and Span80 were in saturated state on the droplet interface at an HLB of 6. At higher HLB values, excess a saturated state on the droplet interface at an HLB of 6. At higher HLB values, excess Tween80 molecules were present and occupied the largest active sites, which led to a lack of further reduction in the droplet size for HLB values greater than 6.0 [24,25]. The mini- mum particle size and PDI were found at the HLB values of 5.0 and 6.0, respectively. Therefore, the HLB values in the 5.0–6.0 range were selected for further experiments. 3.2. Influence of EC The EC was varied in the 4–30 wt% range at a fixed HLB of 5.0. According to the surface tension theory, emulsification occurs by reducing the interfacial tension between the oil and water phases. However, an examination of the data presented in Figure 2 shows that there were no significant differences between the results obtained for the ECs of 4 and 6 wt%. The nanoemulsion gradually became layered below 6 wt% because here the concentration of emulsifier was not sufficient to decrease the interfacial tension, which led to the coalescence of aqueous droplets. At the fraction of 16 wt%, the prepared emul- sion was translucent, whereas at a higher fraction (>16 wt%), the emulsion was more tur- bid. These results indicate that, after the critical micelle concentration was reached, the interfacial tension was not changed by the higher concentration of the surfactant [26]. Appl. Nano 2023, 4 65 Tween80 molecules were present and occupied the largest active sites, which led to a lack of further reduction in the droplet size for HLB values greater than 6.0 [24,25]. The minimum particle size and PDI were found at the HLB values of 5.0 and 6.0, respectively. Therefore, the HLB values in the 5.0–6.0 range were selected for further experiments. 3.2. Influence of EC The EC was varied in the 4–30 wt% range at a fixed HLB of 5.0. According to the surface tension theory, emulsification occurs by reducing the interfacial tension between the oil and water phases. However, an examination of the data presented in Figure 2 shows that there were no significant differences between the results obtained for the ECs of 4 and 6 wt%. The nanoemulsion gradually became layered below 6 wt% because here the concentration of emulsifier was not sufficient to decrease the interfacial tension, which led to the coalescence of aqueous droplets. At the fraction of 16 wt%, the prepared emulsion was Appl. Nano 2023, 4, FOR PEER REVIEW 6 translucent, whereas at a higher fraction (>16 wt%), the emulsion was more turbid. These results indicate that, after the critical micelle concentration was reached, the interfacial tension was not changed by the higher concentration of the surfactant [26]. Figure 2. Effect of EC on the stability of the emulsions. Figure 2. Effect of EC on the stability of the emulsions. Furthermore, an increase in the EC resulted in a decrease in the PDI. As the emulsifier fraction increased from 8 to 16 wt%, the PDI decreased from 0.283 to 0.017, demonstrating Furthermore, an increase in the EC resulted in a decrease in the PDI. As the emulsifier that the formulations prepared at low mass fractions were unstable [14]. fraction increased from 8 to 16 wt%, the PDI decreased from 0.283 to 0.017, demonstrating that the formulations prepared at low mass fractions were unstable [14]. 3.3. Influence of the Oil–Water Ratio (OWR) The oil–water mass ratio will affect the viscosity of the system and the stability of the 3.3. Influence of the Oil–Water Ratio (OWR) emulsion. The oil–water mass ratio was varied in the range from 1:1 to 4:1 at a fixed HLB The oil–water mass ratio will affect the viscosity of the system and the stability of the of 5 and surfactant concentration of 12% (w/w). As shown in Figure 3, it can be observed emulsion. The oil–water mass ratio was varied in the range from 1:1 to 4:1 at a fixed HLB that droplet size decreased from 154.0 nm to 124.0 nm on increasing ratios from 1:1 to of 5 and surfactant concentration of 12% (w/w). As shown in Figure 3, it can be observed 4:1 and the PDI exhibited no big difference. A higher ratio resulted in smaller droplet that droplet size decreased from 154.0 nm to 124.0 nm on increasing ratios from 1:1 to 4:1 size due to decrease in the viscosity of emulsion, and small viscosity is beneficial to the and the PDI exhibited no big difference. A higher ratio resulted in smaller droplet size dispersion of droplets [27]. The emulsions prepared at a ratio of 1:1 to 1.5:1 were found to be unstable after 24 h of storage at room temperature, whereas the emulsions prepared due to decrease in the viscosity of emulsion, and small viscosity is beneficial to the disper- at a ratio of 2:1 to 4:1 were more stable under the same conditions, respectively, and the sion of droplets [27]. The emulsions prepared at a ratio of 1:1 to 1.5:1 were found to be most stable emulsion appeared at the 3:1 oil–water mass ratio. It indicated that emulsions unstable after 24 h of storage at room temperature, whereas the emulsions prepared at a of a higher oil–water ratio reduced the viscosity of the system, leading to dispersing the ratio of 2:1 to 4:1 were more stable under the same conditions, respectively, and the most droplets sufficiently, which could make the droplet size smaller. stable emulsion appeared at the 3:1 oil–water mass ratio. It indicated that emulsions of a higher oil–water ratio reduced the viscosity of the system, leading to dispersing the drop- lets sufficiently, which could make the droplet size smaller. Figure 3. Effects of different OWRs on stability of the emulsions. Appl. Nano 2023, 4, FOR PEER REVIEW 6 Figure 2. Effect of EC on the stability of the emulsions. Furthermore, an increase in the EC resulted in a decrease in the PDI. As the emulsifier fraction increased from 8 to 16 wt%, the PDI decreased from 0.283 to 0.017, demonstrating that the formulations prepared at low mass fractions were unstable [14]. 3.3. Influence of the Oil–Water Ratio (OWR) The oil–water mass ratio will affect the viscosity of the system and the stability of the emulsion. The oil–water mass ratio was varied in the range from 1:1 to 4:1 at a fixed HLB of 5 and surfactant concentration of 12% (w/w). As shown in Figure 3, it can be observed that droplet size decreased from 154.0 nm to 124.0 nm on increasing ratios from 1:1 to 4:1 and the PDI exhibited no big difference. A higher ratio resulted in smaller droplet size due to decrease in the viscosity of emulsion, and small viscosity is beneficial to the disper- sion of droplets [27]. The emulsions prepared at a ratio of 1:1 to 1.5:1 were found to be unstable after 24 h of storage at room temperature, whereas the emulsions prepared at a ratio of 2:1 to 4:1 were more stable under the same conditions, respectively, and the most stable emulsion appeared at the 3:1 oil–water mass ratio. It indicated that emulsions of a Appl. Nano 2023, 4 66 higher oil–water ratio reduced the viscosity of the system, leading to dispersing the drop- lets sufficiently, which could make the droplet size smaller. Appl. Nano 2023, 4, FOR PEER REVIEW 7 Figure 3. Effects of different OWRs on stability of the emulsions. However, under a smaller oil and water ratio, the water content was relatively exces- Figure 3. Effects of different OWRs on stability of the emulsions. sive and additional water led to parts of emulsifiers in the system dispersed in it, which However, under a smaller oil and water ratio, the water content was relatively exces- resulted in insufficient emulsification of liquid paraffin wax, so the particle size of the sive and additional water led to parts of emulsifiers in the system dispersed in it, which emulsion became larger. resulted in insufficient emulsification of liquid paraffin wax, so the particle size of the emulsion became larger. 3.4. Influence of T 3.4. Influence To study ofthe T effects of T, the T was varied in the 30–70 °C range at a fixed HLB of 8.0, EC of 12 wt%, and OWR of 3:1. The T had a significant effect on the droplet size. As shown To study the effects of T, the T was varied in the 30–70 C range at a fixed HLB of in Figure 4, upon increasing the T from 30 to 70 °C, the droplet size increased from 119.36 8.0, EC of 12 wt%, and OWR of 3:1. The T had a significant effect on the droplet size. As to 189.08 nm, and the PDI also increased from 0.015 to 0.035. This indicated that a higher shown in Figure 4, upon increasing the T from 30 to 70 C, the droplet size increased from 119.36 to 189.08 nm, and the PDI also increased from 0.015 to 0.035. This indicated that T influenced the evaporation rate of the organic solvent [28], and the gradual dehydration a higher T influenced the evaporation rate of the organic solvent [28], and the gradual of emulsifier at the oil–water interface gave rise to the increase in interfacial tension, which dehydration of emulsifier at the oil–water interface gave rise to the increase in interfacial promoted the coalescence of liquid droplets [29]. tension, which promoted the coalescence of liquid droplets [29]. Figure 4. Effect of preparation T on stability of the emulsions. Figure 4. Effect of preparation T on stability of the emulsions. 3.5. Orthogonal Experiments Design 3.5. Orthogonal Experiments Design Various methods, such as the single factor method and the response surface method [30], Various methods, such as the single factor method and the response surface method were used to optimize the experimental conditions. In the present study, to reduce the [30], were used to optimize the experimental conditions. In the present study, to reduce experimental time, orthogonal experiments, which can be used to assess the importance the experimental time, orthogonal experiments, which can be used to assess the im- portance of each factor and determine the ideal values, were performed for the optimiza- tion of the preparation conditions of the W/O emulsions [31,32]. In the preparation process of the W/O emulsions, the preparation conditions were optimized by L9 (34) orthogonal experiments using the ADD and the PDI as the assessment indices. The HLB value (A), EC (B), OWR (C), and preparation T (PT (D)) were selected as the experimental factors. Each factor was designed with three levels, and the factors and levels of the orthogonal tests are listed in Table 1, with the results shown in Table 2. Table 1. Factors and levels of Orthogonal experiment. Factor Level HLB EC (wt%) OWR (w/w) T (°C) A B C D 1 5.0 10 2:1 40 2 5.5 12 3:1 50 3 6.0 16 4:1 60 Appl. Nano 2023, 4 67 of each factor and determine the ideal values, were performed for the optimization of the preparation conditions of the W/O emulsions [31,32]. In the preparation process of the W/O emulsions, the preparation conditions were optimized by L9 (34) orthogonal experiments using the ADD and the PDI as the assessment indices. The HLB value (A), EC (B), OWR (C), and preparation T (PT (D)) were selected as the experimental factors. Each factor was designed with three levels, and the factors and levels of the orthogonal tests are listed in Table 1, with the results shown in Table 2. Table 1. Factors and levels of Orthogonal experiment. Factor Level HLB EC (wt%) OWR (w/w) T ( C) A B C D 1 5.0 10 2:1 40 2 5.5 12 3:1 50 3 6.0 16 4:1 60 Table 2. Results of the Orthogonal experiment. HLB EC (wt%) OWR (w/w) T ( C) Run ADD (nm) PDI A B C D 1 1 1 1 1 120.432 0.032 2 1 2 2 2 141.105 0.172 3 1 3 3 3 189.306 0.148 4 2 1 2 3 130.423 0.152 5 2 2 3 1 117.406 0.082 6 2 3 1 2 141.323 0.011 7 3 1 3 2 99.207 0.225 8 3 2 1 3 94.104 0.573 9 3 3 2 1 104.413 0.204 k 150.28 116.69 118.62 114.08 k 129.72 117.54 125.31 127.21 ADD k 99.24 145.01 135.31 137.94 R 51.04 28.33 16.69 23.86 order of importance A > B > D > C optimal level A B D C 3 1 1 1 k 0.12 0.14 0.21 0.11 k 0.08 0.28 0.14 0.14 PDI k 0.33 0.12 0.15 0.29 R 0.25 0.16 0.06 0.19 order of importance A > D > B > C optimal level A D B C 2 1 3 2 As shown in Table 2, the PDI values of runs 7, 8, and 9 were 0.225, 0.573, and 0.204, respectively, while those of the other runs were less than 0.2. The ADD values of runs 7 and 8 were 99.207 and 94.104 nm, respectively, while those of the other runs exceeded 100 nm. The results of the range analysis used to determine the optimal levels of the factors are shown in Table 2. The K/k value for each level was the average of three ADD values and the polydispersity index (PDI), as shown in Table 2. By comparing different K/k values, the optimal level of factors for the preparation of the W/O nanoemulsions could be identified. In this work, the minimum K/k value was better for the optimal level. The difference between the maximum and minimum K/k values is represented by the range value (R), which reflects the detrimental effects of the level on the ADD and PDI. The most important factor was obtained when R was maximal, and the order of im- portance was A > B > D > C and A > D > B > C for the ADD and PDI, respectively [33,34]. Appl. Nano 2023, 4 68 Considering the practicability of the W/O emulsions, the ADD was the most important factor for the formulation selection. The factors influencing the ADD were in the order of HLB value > EC > emulsification T > OWR. The final optimal preparation factors were a HLB of 6.0, EC of 10 wt%, OWR of 2:1, and emulsification T of 40 C. However, the optimal combination was not listed in the nine runs (Table 2), and three additional validation experiments showed that the ADD and the PDI were 45.15 nm and 0.25, respectively, under the optimal preparation conditions (Table 3). These values were smaller than those obtained in any of the nine runs, which confirmed the reliability of the model prediction by the array. Table 3. Confirmatory experiment. Factor HLB EC (wt%) OWR (w/w) T ( C) condition 6 10 2:1 40 times once second third mean Appl. Nano 2023, 4, FOR PEER REVIEW 9 ADD (nm) 73.17 65.91 65.83 45.15 PDI 0.27 0.12 0.3 0.25 Table 3. Confirmatory experiment. Notably, as shown in Figure 5, the laser diffraction measurements exhibited that the W/O nanoemulsion particle size distribution was relatively uniform, and the width was Factor HLB EC (wt%) OWR (w/w) T (°C) substantially unchanged. This indicated that the adjustment of the proportions of the added condition 6 10 2:1 40 materials could significantly reduce the interfacial tension of the water and oil, such that times once second third mean the Ostwald ripening rate was weak, physical changes such as Brownian motion could be ADD (nm) 73.17 65.91 65.83 45.15 resisted, and the nanoemulsion stability could be improved. The more stable the emulsion PDI 0.27 0.12 0.3 0.25 system, the more obvious the protective effect of the gardenia yellow pigment. Figure 5. Particle size distribution of nanoemulsion with the confirmatory experiment. Figure 5. Particle size distribution of nanoemulsion with the confirmatory experiment. 3.6. Rheology 3.6. Rheology 3.6.1. Shear Flow Tests 3.6.1. Shear Flow Tests Research has revealed that high emulsion viscosity is conducive to its stability [35]. As Research has revealed that high emulsion viscosity is conducive to its stability [35]. shown in Figure 6a, the viscosity decreased significantly with the increase in the shear rate. As shown in Figure 6a, the viscosity decreased significantly with the increase in the shear At higher shear rates, the viscosity tended to be almost constant, indicating that the W/O rate. At higher shear rates, the viscosity tended to be almost constant, indicating that the nanoemulsion was a pseudoplastic fluid with shear thinning behavior [36]. This was due W/O nanoemulsion was a pseudoplastic fluid with shear thinning behavior [36]. This was to the higher shear strain leading to more obvious elongation, deformation, and network due to the higher shear strain leading to more obvious elongation, deformation, and net- fracture of the nanoemulsion droplets [37]. work fracture of the nanoemulsion droplets [37]. (a) Appl. Nano 2023, 4, FOR PEER REVIEW 9 Table 3. Confirmatory experiment. Factor HLB EC (wt%) OWR (w/w) T (°C) condition 6 10 2:1 40 times once second third mean ADD (nm) 73.17 65.91 65.83 45.15 PDI 0.27 0.12 0.3 0.25 Figure 5. Particle size distribution of nanoemulsion with the confirmatory experiment. 3.6. Rheology 3.6.1. Shear Flow Tests Research has revealed that high emulsion viscosity is conducive to its stability [35]. As shown in Figure 6a, the viscosity decreased significantly with the increase in the shear rate. At higher shear rates, the viscosity tended to be almost constant, indicating that the W/O nanoemulsion was a pseudoplastic fluid with shear thinning behavior [36]. This was due to the higher shear strain leading to more obvious elongation, deformation, and net- Appl. Nano 2023, 4 69 work fracture of the nanoemulsion droplets [37]. Appl. Nano 2023, 4, FOR PEER REVIEW 10 (a) (b) Figure 6. The relationship between viscosity and shear rate for optimal nanoemulsion (a); The Figure 6. The relationship between viscosity and shear rate for optimal nanoemulsion (a); The change change of elastic modulus and viscous modulus with frequency (b). of elastic modulus and viscous modulus with frequency (b). 3.6.2. Oscillation Strain Sweep 3.6.2. Oscillation Strain Sweep Oscillation strain sweep tests were conducted to characterize the structural strengths of Oscillation strain sweep tests were conducted to characterize the structural strengths of the emulsions in the linear viscoelastic region. Figure 6b shows the frequency dependence the emulsions in the linear viscoelastic region. Figure 6b shows the frequency dependence of the dynamic modulus. The storage modulus (G′) is a measure of the energy stored elas- of the dynamic modulus. The storage modulus (G ) is a measure of the energy stored tically during an oscillation cycle, whereas the loss modulus (G″) is a measure of the energy elastically during an oscillation cycle, whereas the loss modulus (G”) is a measure of the dissipated by the viscous flow during the oscillation. The G′ modulus was negligible com- energy dissipated by the viscous flow during the oscillation. The G modulus was negligible pacompar red to the ed G to″the for t G” he for nanoemu the nanoemulsions lsions with 1 with 0% 10% (w/w()w s/ uw rf)ac surfactant tant concentr concentrations; ations; thus, thus, the emulsions exhibited a predominant viscous behavior. the emulsions exhibited a predominant viscous behavior. However However, , as as can can be be s seen een fr from om Figur Figure e 7 7, , when when th the e surfactant surfactant concentration w concentration was as 20% 20% and and 2 24% 4% ( (w w/ /w w), ), the the G G′ v value alue o of f the the two two em emulsions ulsions w was as ab about out twic twice e as as high high as as t that hat o of f G G”,″, indicating that the emulsion had a certain elasticity. The modulus of elasticity depended on indicating that the emulsion had a certain elasticity. The modulus of elasticity depended the types of interaction forces between the droplets and their nearest neighbors—attractive on the types of interaction forces between the droplets and their nearest neighbors—at- or trar cepulsive tive or rep [38 uls ,39 iv]. e [ The 38,39 change ]. The change from a pr from edominantl a predom y iviscous nantly vto iscou a pr s to edominant a predom lyinan elastic tly elastic response was affected by the ratio of the adsorption thickness of the surfactant to the droplet radius R [39]. Appl. Nano 2023, 4 70 Appl. Nano 2023, 4, FOR PEER REVIEW 11 response was affected by the ratio of the adsorption thickness of the surfactant to the droplet radius R [39]. (a) (b) Figure 7. dynamic (a,b) rheological analyses of W/O emulsions with different ECs. (a) dynamic rhe- Figure 7. Dynamic (a,b) rheological analyses of W/O emulsions with different ECs. (a) dynamic ological analysis of W/O emulsions with different ECs and elastic moduli G′, and (b) dynamic rhe- rheological analysis of W/O emulsions with different ECs and elastic moduli G , and (b) dynamic ological analysis of W/O emulsions with different ECs and loss moduli G″. rheological analysis of W/O emulsions with different ECs and loss moduli G”. 3.7. Stability Evaluation of the Emulsions 3.7. Stability Evaluation of the Emulsions In this study, the effects of different chloride salts (sodium, potassium, aluminum, In this study, the effects of different chloride salts (sodium, potassium, aluminum, and and calcium) at 0.1 M concentrations on the long-term stability of the W/O nanoemulsions calcium) at 0.1 M concentrations on the long-term stability of the W/O nanoemulsions were were examined. As shown in Figure 8, the addition of chloride salts in the aqueous phase examined. As shown in Figure 8, the addition of chloride salts in the aqueous phase had had little effect on the initial ADD and did not result in significant differences in the sta- little effect on the initial ADD and did not result in significant differences in the stability bility compared to the systems with no added salt. This indicated that the EC was the compared to the systems with no added salt. This indicated that the EC was the main main determinant of the initial particle size of the W/O nanoemulsions. As shown in Fig- determinant of the initial particle size of the W/O nanoemulsions. As shown in Figure 9, ure 9, after 30 days of observation at room T, the emulsions containing pure water or 0.1 after 30 days of observation at room T, the emulsions containing pure water or 0.1 M CaCl M CaCl2 and AlCl3 slightly separated, whereas the emulsions containing 0.1 M KCl and and AlCl slightly separated, whereas the emulsions containing 0.1 M KCl and NaCl did NaCl did not show any separation until 30 days at room T. This difference was related to the number of valence electrons of the cations of these salts. Appl. Nano 2023, 4 71 Appl. Nano 2023, 4, FOR PEER REVIEW 12 Appl. Nano 2023, 4, FOR PEER REVIEW 12 not show any separation until 30 days at room T. This difference was related to the number of valence electrons of the cations of these salts. Figure 8. Long-term stability of pure water nanoemulsions and nanoemulsions with 0.1 M salts. Figure 8. Long-term stability of pure water nanoemulsions and nanoemulsions with 0.1 M salts. Figure 8. Long-term stability of pure water nanoemulsions and nanoemulsions with 0.1 M salts. Figure 9. Appearance of pure water nanoemulsions and nanoemulsions with 0.1 M salts after stor- Figure 9. Appearance of pure water nanoemulsions and nanoemulsions with 0.1 M salts after stor- Figure 9. Appearance of pure water nanoemulsions and nanoemulsions with 0.1 M salts after storage age for 30 days. The type of salt from left to right are pure water, AlCl3, CaCl2, NaCl, and KCl. age for 30 days. The type of salt from left to right are pure water, AlCl3, CaCl2, NaCl, and KCl. for 30 days. The type of salt from left to right are pure water, AlCl , CaCl , NaCl, and KCl. 3 2 KCl and NaCl are ionic compounds, and upon addition to the water phase, these KCl KCl and and NaCl NaCl are are ionic ionic compounds, compounds, a and nd upon upon add addition ition to to the the wate waterr phase, phase, these these + + + + − + + − crystals dissociate into Na , K , and Cl ions in water. It is well known that the emulsifica- crystals dissociate into Na , K , and Cl ions in water. It is well known that the emul- crystals dissociate into Na , K , and Cl ions in water. It is well known that the emulsifica- tion process includes the break-up of droplets and the re-coalescence of the newly formed sification process includes the break-up of droplets and the re-coalescence of the newly tion process includes the break-up of droplets and the re-coalescence of the newly formed droplets. The investigations by Jafari et al. [40]. showed that the reduction in the re-coa- formed droplets. The droplets. invest The ig inv ation estigations s by Jafari e by tJafari al. [40]. show et al. [40]. ed th showed at the re that duction in the reduction the re-co in the a- lescence w re-coalescence as strong was str er th onger an the enhanc than the enhancement ement of of the the drop droplet let ddisr isrup uption. tion. Acco Accor rding ding to to lescence was stronger than the enhancement of the droplet disruption. According to T Ta adr dros os [ [41 41], ], t the he s stability tability of of the the oi oil–water l–water in interface terface dep depends ends on on the the e electr lectros ostatic tatic or or sp spatial atial Tadros [41], the stability of the oil–water interface depends on the electrostatic or spatial repul repulsion sion in interact teractions ions. . The Ther refore, efore, when when KCl KCl and and N NaCl aCl d dissociated issociated iinto nto ions ions in in w water ater,, due due repulsion interactions. Therefore, when KCl and NaCl dissociated into ions in water, due to to the the re reduced duced re re-coalescence, -coalescence, the these se ion ions s g gave ave r rise ise to to repul repulsive sive in interactions teractions in in th the e drop droplets lets to the reduced re-coalescence, these ions gave rise to repulsive interactions in the droplets that could improve the stability of the oil–water interface. This conclusion agrees with the that could improve the stability of the oil–water interface. This conclusion agrees with the that could improve the stability of the oil–water interface. This conclusion agrees with the results reported by Scherze et al. [42]. results reported by Scherze et al. [42]. results reported by Scherze et al. [42]. For the effect of CaCl and AlCl on the nanoemulsion stability, it was suggested For the effect of CaCl2 and 2 AlCl3 on the nanoemu 3 lsion stability, it was suggested that For the effect of CaCl2 and AlCl3 on the nanoemulsion stability, it was suggested that that a reduction in the attractive force between the water droplets due to the addition of a reduction in the attractive force between the water droplets due to the addition of the a reduction in the attractive force between the water droplets due to the addition of the the electrolytes in the aqueous phase increased the W/O emulsion stability with respect electrolytes in the aqueous phase increased the W/O emulsion stability with respect to the electrolytes in the aqueous phase increased the W/O emulsion stability with respect to the to the coalescence. Consequently, the attractive forces were minimized by matching the coalescence. Consequently, the attractive forces were minimized by matching the refrac- coalescence. Consequently, the attractive forces were minimized by matching the refrac- refractive indices and/or the dielectric constants of the two phases. As the electrolyte tive indices and/or the dielectric constants of the two phases. As the electrolyte concentra- tive indices and/or the dielectric constants of the two phases. As the electrolyte concentra- concentration increased, the refractive index of the aqueous phase increased, and the tion increased, the refractive index of the aqueous phase increased, and the ionization tion increased, the refractive index of the aqueous phase increased, and the ionization ionization constant decreased [43]. Thus, the addition of calcium and aluminum salts constant decreased [43]. Thus, the addition of calcium and aluminum salts decreased the constant decreased [43]. Thus, the addition of calcium and aluminum salts decreased the decreased the attractive forces, leading to the reduced frequency of collisions between attractive forces, leading to the reduced frequency of collisions between the water droplets, attractive forces, leading to the reduced frequency of collisions between the water droplets, the water droplets, improving the stability with respect to the coalescence of the W/O improving the stability with respect to the coalescence of the W/O emulsions, and leading improving the stability with respect to the coalescence of the W/O emulsions, and leading emulsions, and leading to their sedimentation. to their sedimentation. to their sedimentation. Appl. Nano 2023, 4 72 The above results demonstrated that the spatial electrostatic effects of the monovalent cationic (KCl and NaCl) and the nonionic surfactants were greater than the delamina- tion/sedimentation forces of the system, providing effective resistance to the destabilizing processes, such as Ostwald ripening, coalescence, and flocculation. Thus, the emulsions with NaCl and KCl had small particle sizes, narrow particle size distributions, and better long-term stability, whereas the nanoemulsions with salts based on cations with valences greater than one (CaCl and AlCl ) showed the opposite effects. 2 3 4. Conclusions In this work, gardenia-yellow-pigment-based W/O nanoemulsions were produced, and the effect of the surfactant and metal ions on the nanoemulsions was studied. The optimal W/O nanoemulsions’ preparation parameters were defined by single factor experi- ments and orthogonal tests, including the hydrophilic–lipophilic balance (HLB) value (6), the emulsifier concentration (10%), the oil–water ratio (2:1), and the preparation tempera- ture (40 C). The experimental analysis showed that the W/O nanoemulsions were shear thinning pseudoplastic fluids, and the rheology of the W/O nanoemulsions changed from viscosity to elasticity by adding the appropriate concentration of surfactant. In particular, the stability of the nanoemulsion was improved by electrostatic and spatial exclusion after NaCl and KCl addition, whereas the instability mechanism after CaCl and AlCl addition 2 3 resulted in coalescence and sedimentation. This preliminary study provides new insight into the design of new generation nanoemulsions containing gardenia yellow pigment for health promotion. The outcomes can be considered as the beginning, requiring further research focused on gardenia yellow pigment for commercial usage. Author Contributions: Conceptualization, L.G. and B.L.; methodology, L.G.; software, L.G.; valida- tion, L.G. and B.L.; formal analysis, L.G.; investigation, L.G.; resources, L.G. and B.L.; data curation, L.G.; writing—original draft preparation, L.G.; writing—review and editing, L.G. and B.L.; visualiza- tion, L.G.; supervision, L.G.; project administration, L.G.; funding acquisition, B.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was financially supported by the Hubei Provincial Natural Science Foundation major project (2009CDA102) from Hubei Province, China and the APC was funded by [2009CDA102]. Data Availability Statement: The data presented in this study are available from the authors. Acknowledgments: Li Gao and Bin Li contributed equally to this work. 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Journal

Applied Nano – Multidisciplinary Digital Publishing Institute

Published: Apr 4, 2023

Keywords: gardenia yellow pigment; nanoemulsion; hydrophilic–lipophilic balance; stability; rheology

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