A Comparative Study of O/W Nanoemulsions Using Extra Virgin Olive or Olive‐Pomace Oil: Impacts on Formation and Stability
Abstract: Nanoemulsions are considered an innovative approach for industrial food applications. The present study explored the potential use of olive-pomace oil (OPO) for oil-in-water (o/w) nanoemulsion preparations and compared the effectiveness of extra virgin olive oil (EVOO) and OPO at nanoemulsion formulations. The ternary-phase diagrams were constructed and the o/w nanoemulsions properties were evaluated in relation to their composition. The results showed that it is possible to form OPO nanoemulsions using Polyso… Show more
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Olive pomace, the solid by-product derived from olive oil production consists of a high concentration of bioactive compounds with antioxidant activity, such as phenolic compounds, and their recovery by applying innovative techniques is a great opportunity and challenge for the olive oil industry. This study aimed to point out a new approach for the integrated valorization of olive pomace by extracting the phenolic compounds and protecting them by encapsulation or incorporation in nanoemulsions. Innovative assisted extraction methods were evaluated such as microwave (MAE), homogenization (HAE), ultrasound (UAE), and high hydrostatic pressure (HHPAE) using various solvent systems including ethanol, methanol, and natural deep eutectic solvents (NADESs). The best extraction efficiency of phenolic compounds was achieved by using NADES as extraction solvent and in particular the mixture choline chloride-caffeic acid (CCA) and choline chloride-lactic acid (CLA); by HAE at 60 °C/12,000 rpm and UAE at 60 °C, the total phenolic content (TPC) of extracts was 34.08 mg gallic acid (GA)/g dw and 20.14 mg GA/g dw for CCA, and by MAE at 60 °C and HHPAE at 600 MPa/10 min, the TPC was 29.57 mg GA/g dw and 25.96 mg GA/g dw for CLA. HAE proved to be the best method for the extraction of phenolic compounds from olive pomace. Microencapsulation and nanoemulsion formulations were also reviewed for the protection of the phenolic compounds extracted from olive pomace. Both encapsulation techniques exhibited satisfactory results in terms of encapsulation stability. Thus, they can be proposed as an excellent technique to incorporate phenolic compounds into food products in order to enhance both their antioxidative stability and nutritional value.
Olive pomace, the solid by-product derived from olive oil production consists of a high concentration of bioactive compounds with antioxidant activity, such as phenolic compounds, and their recovery by applying innovative techniques is a great opportunity and challenge for the olive oil industry. This study aimed to point out a new approach for the integrated valorization of olive pomace by extracting the phenolic compounds and protecting them by encapsulation or incorporation in nanoemulsions. Innovative assisted extraction methods were evaluated such as microwave (MAE), homogenization (HAE), ultrasound (UAE), and high hydrostatic pressure (HHPAE) using various solvent systems including ethanol, methanol, and natural deep eutectic solvents (NADESs). The best extraction efficiency of phenolic compounds was achieved by using NADES as extraction solvent and in particular the mixture choline chloride-caffeic acid (CCA) and choline chloride-lactic acid (CLA); by HAE at 60 °C/12,000 rpm and UAE at 60 °C, the total phenolic content (TPC) of extracts was 34.08 mg gallic acid (GA)/g dw and 20.14 mg GA/g dw for CCA, and by MAE at 60 °C and HHPAE at 600 MPa/10 min, the TPC was 29.57 mg GA/g dw and 25.96 mg GA/g dw for CLA. HAE proved to be the best method for the extraction of phenolic compounds from olive pomace. Microencapsulation and nanoemulsion formulations were also reviewed for the protection of the phenolic compounds extracted from olive pomace. Both encapsulation techniques exhibited satisfactory results in terms of encapsulation stability. Thus, they can be proposed as an excellent technique to incorporate phenolic compounds into food products in order to enhance both their antioxidative stability and nutritional value.
Development of extra virgin olive and olive pomace oil nanoemulsions (o/w and w/o) enriched with surface‐active phenolic compounds
The present study explored the potential use and the effectiveness of extra virgin olive oil (EVOO) and olive pomace oil (OPO) for water in oil (w/o) and oil in water (o/w) nanoemulsion preparations. Nonionic food grade emulsifiers were examined in the formulation of the nanoemulsions and the incorporation of polyphenols extracted from olive kernel on their physicochemical properties was also investigated. The effect of the dispersed phase and the incorporated bioactive compounds was evaluated based on the mean droplet diameter (MDD) and polydispersity index (PDI), turbidity and viscosity, MDD growth, surface tension, and total phenolic content of the nanoemulsions during storage. Results were statistically evaluated using analysis of variance and Duncan's test was performed where applicable. The most effective type and the optimum ratio of the emulsifier was determined in each system (o/w–w/o, EVOO–OPO). The nanoemulsions produced presented desirable properties, in terms of kinetic stability, MDD, PDI, and viscosity and they demonstrated satisfactory physical and chemical stability during storage. MDD and PDI values were minimum in the case of o/w nanoemulsion prepared with 4% Tween 40. The stability of the nanoemulsion was enhanced by polyphenols while their retention reached ~80% being higher in o/w nanoemulsions. It was concluded that it is possible to prepare fine o/w and w/o nanoemulsions using both EVOO and OPO. The incorporation of bioactive compounds can significantly affect the nanoemulsions' properties, improving their kinetic and chemical stability. Practical Applications Although olive oil has been thoroughly studied for nanoemulsion preparations, there is still limited study on olive pomace oil applications. Hence, this present study was designed to compare the effectiveness of extra virgin olive oil and olive pomace oil on the formation of o/w and w/o nanoemulsion enriched with phenolic compounds extracted from the olive kernel. The novelty of work is highlighted in the following: both extra virgin olive and olive pomace oil can be suitable for the preparation of o/w and w/o nanoemulsions with good physical stability and without excessive droplet growth upon storage. These nanoemulsions can be used as a suitable means for the incorporation and protection of bioactive ingredients, such as polyphenolic compounds, and serve as an excellent delivery system as they exhibit satisfactory retention during storage.
The new trend in food industry has been focused on fortification of foods with health-promoting ingredients (nutrients and nutraceuticals) specifically designed to improve human health and well-being. Many of these bioactive components categorized either as hydrophilic (vitamins, minerals, polyphenols) or lipophilic components (polyunsaturated lipids, oil-soluble vitamins, phytosterols, curcuminoids, carotenoids, and flavonoids). Thus, many food-grade delivery systems have been developed (nanoemulsions, liposomes, nanoparticles) to incorporate active ingredient. Nanosized structures such as nanoemulsions are regarded as useful tools with great potential in the food sector for the delivery of food ingredients. Moreover, in the last decade, the introduction of more efficient emulsification technologies and highly surface-active emulsifiers facilitated the large-scale production of nanoemulsions. Nanoemulsions are metastable colloidal dispersions, consisting of one liquid being dispersed in the form of small spherical droplets to another immiscible liquid. They are thermodynamically unstable systems but kinetically stable presenting high resistance to structural changes through time such as coalescence, flocculation, and gravitational separation. Nowadays, nanoemulsions are gaining increased interest as delivery and encapsulation systems for various bioactive ingredients, due to their unique functional characteristics and physicochemical properties, such as high physical stability and optical clarity/transparent appearance as well as enhanced bioavailability. Due to the abovementioned properties, they are considered as excellent delivery systems controlling the quality, the flavor, and the antimicrobial properties of products and also the bioavailability of the incorporated compound. Double nanoemulsions are complex polydispersed multiphase systems consisting of a nanoemulsion dispersed in a continuous phase. Most studies focus on water-in-oil-in-water multiple nanoemulsions rather than oil-in-water-in-oil (o1/w/o2) type. Multiple o1/w/o2nanoemulsions presents various advantages as delivery systems due to their compartmentalized structure, as they can simultaneously deliver both oil-soluble and water-soluble compounds, while protecting them against chemical degradation. Despite their potential in product development, the production of o1/w/o2multiple nanoemulsions is practically limited due to their increased instability. Double emulsions are often produced by a two-step emulsification method using various high-shear devices, high pressure- homogenizers, microfluidizers and membrane systems. The formation of the final double nanoemulsion using ultrasound-assisted two-step homogenization process gain more a more as the ultrasonic device is more effective producing small droplets at less process time. The stability of multiple emulsions is influenced by their composition and emulsification conditions. The first step is to prepare an inner phase with very small droplets preventing, while in the second step the first emulsion should be dispersed in a second continuous phase with a high yield of inner droplets and with smaller external droplets. Nowadays, there has been a growing demand to fortify foods with health-promoting ingredients such as conjugated linoleic acid (CLA) or coenzyme Q10 (CoQ10) using food-delivery systems. CLA is gaining recognition as a food supplement due to its significant physiological activities. CoQ10 which is an endogenous essential molecule for the human body is also known as an excellent antioxidant compound. However, these lipophilic bioactive compounds, due to their physical or chemical instability or water insolubility, cannot simply be incorporated into these products. The nanoemulsions, in order to be effective delivery systems, should maintain their droplet size, be resistant to creaming while they should protect the incorporated bioactive ingredients during shelf-life (retention percent). Given the above, designing nanoemulsions and multiple nanoemulsions with long-term shelf-life under refrigerated and environmental storage is an essential goal for industries. The aim of this study was to investigate and understand the effect of the destabilization phenomena during manufacture, storage, and the affecting factors of these systems such as the ratio of dispersed and continuous phase, the composition of the oil phase, the type of emulsifiers, the droplet size and distribution of dispersed phase and the potential presence of compounds with emulsifying and surface-active characteristics. The nature of the lipid phase plays crucial role determining the droplet size, the viscosity, the stability and the interfacial tension of the emulsions. Various lipid types have already been examined in multiple emulsions production, however, there are limited studies on extra virgin olive (EVOO) and olive pomace oil (OPO) as lipid phase. The selection of the lipid phase can serve as a strategy to favor functional and health promoting food products. Both vegetable oils, due to their medium-chain triglycerides content, are not prone to oxidation while it is considered to be excellent for applications involving high temperatures. Thus, initially, the present study focuses on the potential use of extra virgin olive and olive-pomace oil for the preparation of oil-in-water (o/w) nanoemulsions fortified with CLA or CoQ10 and the effect of the oil fraction and emulsifier types on nanoemulsion’s properties were investigated. It was investigated the effect of environment (25 °C) and refrigerated (4 °C) storage on the nanoemulsion physical and chemical stability. Meanwhile, it was established the most stable o/w nanoemulsion system which was used as the dispersed phase for the o1/w/o2nanoemulsions. Moreover, the surface tension characteristics of the non-ionic emulsifiers and their mixture were examined in order to determine the optimum emulsifier concentration for the multiple nanoemulsions. Their stability was investigated under different processing conditions using low and high HLB emulsifiers, and examining different dispersed phases and dispersed phase volume fraction of the primary o/w nanoemulsion. Finally, it was investigated the addition of phenolic compounds, extracted from olive kernel, in the aqueous phase of the o1/w/o2nanoemulsions. The alterations in their physico-chemical properties during storage were examined by monitoring mean droplet diameter (MDD), the emulsion stability index (ESI%), encapsulation efficiency (oiling off% and EE%) bioactive compounds retention, total phenolic content (TPC) and antioxidant radical scavenging (DPPH). The present study explored the potential use of olive-pomace oil for oil-in-water nanoemulsions and compared the effectiveness of EVOO and OPO at nanoemulsion formulations. Concluding, it can be confirmed that o/w nanoemulsions with extra virgin olive and olive-pomace oil and various emulsifiers’ concentrations presented desirable properties, in terms of kinetic stability, droplet size and size distribution (PDI), ζ-potential, viscosity and turbidity. The ternary-phase diagrams were constructed and the o/w nanoemulsions properties were evaluated in relation to their composition. The results showed that it is possible to form OPO nanoemulsions using Tween 20 or Tween 40. EVOO exhibited lower surface and interfacial tension forming nanoemulsions with a high ESI% and a low MDD. However, OPO led to nanoemulsions with a high ESI% but with a higher MDD. It was observed that by increasing the emulsifier concentration the MDD decreased, while increasing the dispersed phase concentration led to a higher MDD and a lower ESI%. Finally, nanoemulsions with the smallest MDD (99.26 ± 4.20 nm) and PDI (0.236 ± 0.010) were formed using Tween 40, which presented lower surface and interfacial tension. Specifically, the nanoemulsion with 6 wt% EVOO and 6 wt% Tween 40 demonstrated an interfacial tension of 51.014 ± 0.919 mN m−1 and with 6 wt% OPO and 8 wt% Tween 20 presented an MDD of 99.26 ± 4.20 nm. However, the nanoemulsion interfacial tension of 54.308 ± 0.089 mN m−1 and an MDD of 340.5 ± 7.1 nm.In addition, CoQ10 nanoemulsions were prepared using extra virgin olive or olive-pomace oil with Tween 20 and Tween 40. The results showed that it is possible to form fine o/w nanoemulsions with the two oils and various emulsifiers’ concentrations. Moreover, both lipid phases resulted in CoQ10-loaded nanoemulsions with high physical and chemical stability under different storage conditions, while extra virgin olive oil further protect CoQ10exhibiting higher retention. All examined conditions led to o/w nanoemulsions with droplet size in the nano range, narrow droplet size distribution, satisfactory droplet charge transparent appearance, and high chemical stability (RCoQ10%). EVOO proved able to form kinetically more stable nanoemulsions with high CoQ10 retention (74.01%), while OPO led to nanosized emulsions with lower CoQ10 retention (71.99%). It was also observed that CoQ10 retention increases as the oil concentration increases (6% w/w EVOO, RCoQ10 = 77.17% and 8% w/w EVOO, RCoQ10 = 79.89%). All the nanoemulsion formulations, after storage either at 4 °C or at 25 °C, remained in the nanosized range after 3 months, with high physical (MDD < 500 nm) and chemical stability (RCoQ10 = 52.87%). Nanoemulsions with 4% w/w emulsifier concentration appeared kinetically and chemically more stable as they presented lower MDD variations during storage. Thus, oil-in-water nanoemulsions are considered excellent delivery systems for CoQ10, offering high protection and controlled release of the bioactive compoundLikewise, oil-in-water nanoemulsions based on extra virgin olive or pomace oil are considered excellent delivery systems offering high protection and controlled release of CLA. All formulations demonstrated good long-term physical and chemical stability; mean droplet diameter less than 300 nm during 60 days-storage (4°C and 25°C). Specifically, CLA degradation (RCLA %) was lower in the nanoemulsions with 8% wt lipid phase; specifically, nanoemulsions with 8% wt olive pomace oil and 6% wt Tween 20 (RCLA=80.154% at 25 oC and RCLA=84.165% 4 oC) after 2 months. Moreover, CLA retention was significantly affected by the type and the ratio of the emulsifier. The retention was higher using 6% wt Tween 40 (73.06%). It should be highlighted that these nanoemulsions with 6% wt emulsifier concentration presented also lower MDD variations during storage. Response surface methodology was used to optimize the preparation conditions (type and concentration of lipid phase and emulsifier) at different storage conditions and predict the optimum values of mean droplet diameter, ζ-potential and CLA retention. The minimum lipid concentration (4% wt) exhibited the optimum values at both environment and refrigerated storage for EVOO nanoemulsions. However, nanoemulsion with 8% wt OPO and 4 % wt Tween 40 (71.843% at 25 oC) or 6% wt Tween 20 were the optimum condition regarding the highest chemical stability. After establishing the optimum conditions for the formation of nanoemulsions with good physical (limited MDD growth) and chemical stability (limited deterioration of the incorporated compounds), the study focused on the preparation of oil-in-water-in-oil (o1/w/o2) multiple nanoemulsions. At the following section, the dispersed phases of the o1/w/o2 nanoemulsions were comparatively evaluated in order to investigate their effect on the particle size (MDD), droplet size distribution (PDI), gravitational phase separation (ESI%), encapsulation (oiling off%, EE%) and chemical stability of the multiple emulsions. Τhe study evaluated the key parameters controlling the stability and functionality of the double nanoemulsions. The surface tension characteristics of the non-ionic emulsifiers (Span 20, Span 80, Tween 20, Tween 40 and Tween 80) and their mixture were examined in order to determine the optimum emulsifier concentration. Oil-in-water-in-oil nanoemulsions were produced with nonionic emulsifiers by combining high speed and ultrasonic homogenizer. The surface activity of the emulsifiers is considered an important parameter, surface tension measurements at air/oil interface can elucidate the surface activity of the non-ionic emulsifiers and thus find their minimum adequate concentration. It was observed that the air/EVOO surface tension was affected by both emulsifier type and its concentration. Generally, the surface tension decreased above concentration 2% wt, however a slight increase was observed over concentration 8% wt. The lowering of the surface tension was greater for the Span 20, particularly at 4% wt; also Span 80 exhibited low ST values at concentration above 4% wt. However, even at lower concentration (1% wt), all emulsifiers reduced the surface tension of bulk EVOO (31.966 ± 0.099 mN/m). Multiple nanoemulsions with desirable properties in terms of particle size, stability, viscosity, encapsulation efficiency and microstructure were obtained by ultrasonic homogenization for 120 -180 s. Most of the double emulsions showed a bimodal particle size distribution, (PDI= 0.471 ± 0.033), regardless of the type and the concentration of the emulsifier used. This appearance is typical for o1/w/o2-type double emulsions prepared with a two-step procedure. The lowest concentrations (1% and 2% wt) of each emulsifier were not very effective as they tend to form big particles. The lowest mean droplet diameter was exhibited in the double emulsions with 6E6T40 (6% wt Tween 40, 6% wt EVOO and 88% wt water) nanoemulsion as a dispersed phase, this dispersed phase presented the lowest droplet size, surface tension and viscosity compared to others. Increasing the dispersed volume fraction, resulted in higher particle size, gravitational separation and high oiling off% values. Regarding the emulsifier type, Span 20, Span 80, Span 80: Tween 20, Span 80: Tween 40 and Span 80: Tween 80 presented the lowest surface tension and facilitated the homogenization resulting in double emulsions with MDD = 528.35 ± 29.10 nm. Respectively, the emulsifiers with high surface tension (Tween 20, Tween 40 and Tween 80) formed double emulsions with proportionally high MDD = 739.92 ± 91.04 nm. Although, no significant differences were observed in the viscosity of the double emulsions, only a slight increase was observed by increasing the emulsifier’s concentration, while the viscosity values of o1/w/o2emulsions processed for t=180 s (89.05 ± 3.15cP) were significantly lower than those processed for t=120 s (92.89 ± 4.03cP). Double nanoemulsions with high viscosity also exhibited high MDD values. The majority of the double emulsions had a transparent appearance with turbidity values varying from 111 NTU to 605 NTU, while several samples were opaque with values higher than 700 NTU. In particular, Span 80 plain and/or in mixture induced lower turbidity values. However, emulsifiers such as Span 80 and Span 80: Tween 40, at 8% wt concentration produced turbid samples (i.e. 8% wt S80:972 NTU, 8% wt S80T40: 559 NTU). As far as the emulsion stability index (ESI%) is concerned after 4 week-storage at 25°C, certain samples with emulsifier Span 20 or Span 80 presented high physical stability without phase separation. However, Tween emulsifiers led to double emulsions with low kinetic stability (ESI%<85%). Moreover, the lower ultrasonic treatment resulted in higher ESI% values, indicating that ESI% and MDD analyses were very consistent. The dispersed phase significantly affected the ESI%. In particular, the double emulsion with the o/w nanoemulsion 8E8T20 (8% wt Tween 20, 8% wt EVOO and 84% wt water) as dispersed phase continuously decreased during storage. However, double emulsions with the nanoemulsion 4E4T20 (4% wt Tween 20, 4% wt EVOO and 92% wt water) or 6E6T40 as dispersed phase presented an appreciable decrease only at the last week of storage.The dispersed volume fractions of the o1/w/o2 nanoemulsions were compared in order to investigate their effect on the particle size, droplet size distribution and gravitational phase separation of the double emulsions. The mean droplet diameter of the double o1/w/o2 emulsions as a function of the volume fraction of o/w nanoemulsions for the different experimental conditions (emulsifier type, ultrasound process time, o/w composition) ranged from 288 nm to 1297 nm. It was found that the highest volume fraction resulted in the largest MDD (851.20 ± 17.15 nm), also, increasing the dispersed phase volume (o1/w) the MDD of double o1/w/o2 emulsions increased. Moreover, the type of dispersed phase (type of o/w nanoemulsion) significantly affected the mean droplet diameter, with the nanoemulsion 6E6T40 resulted to o1/w/o2nanoemulsions presenting the minimum MDD (611.05 ± 22.50 nm). The ultrasonic process time did not significantly influence the MDD, although the combination of higher process time (180 s) and the medium volume fraction (5% wt) of the dispersed phase 6E6T40 resulted in the lowest MDD (447.16 ± 8.42nm). In addition, the emulsifier type significantly affected the droplet size, specifically Span 20 and the mixture Span20/Tween 40 producing double o1/w/o2emulsion with lower MDD (range from 625.27 to 643.20 nm). Surprisingly, it was observed that certain formulations with MDD < 500 nm exhibited high kinetic stability with no phase separation or creaming during storage. However, double emulsions with MDD values near to 0.1μm presented rapidly creaming phenomena and consequently phase separation. Moreover, it was found that the samples with higher emulsifier concentration and dispersed volume fraction had the maximum viscosity values however, their droplet size remained relatively stable in long term with limited phase separation. The examined emulsifiers did not differ significantly in terms of ESI%; double emulsions with Span 20: Tween 40 or Tween 40 presented lower ESI% values. Moreover, it was observed that the double emulsion with the 4E4T20 as inner phase had high ESI% values. Encapsulation efficiency is an important parameter concerning double emulsion, correlating with their droplet size and physical stability. It was found that the concentration of Sudan Red III could not be fully measured on the majority of the double emulsions; indicating that the marker preferentially remained with the inner lipid phase. Regarding the examined conditions, only the dispersed volume fraction affected significantly the recovery of the dyed oil. Specifically, the double emulsion with 4E4T20 as inner phase presenting the lowest oiling off% values. Similar results were observed between the kinetic stability (ESI%) and oiling off% values. The double emulsion with high stability did not present leak of dyed oil phase (o1) in the external phase (o2). The oiling off values above 15%, are consistent with the visual appearance of the double emulsions which exhibited phase separation.In addition, differential scanning calorimetry (DSC) was employed in order to confirm evaluate the encapsulation efficiency and also the complex structure of the double nanoemulsions. The DSC thermographs can give some qualitative information about the droplet size distribution of the inner dispersed phase and the extent of destabilization mechanisms (phase separation). High encapsulation stability yield was observed indicating the successful use of ultrasound to the production of double nanoemulsions. It should be noted that the intensity of the ultrasound did not destroy the double structure of the colloid system. Moreover, it was found that the double nanoemulsions with the low mean droplet diameter presented also low encapsulation yield (EE%). For instance, the sample with a mean droplet diameter of 432 ± 5.15 nm also exhibited EE%= 92.399% and ESI% = 90%. On the other hand, samples with mean droplet diameter of 1155.73 ± 25.19 nm had higher encapsulation yield (EE%= 98.53%) and the ESI% values were 93.3%. The DSC thermograms showed also differences in the structure of the double emulsions. For instance, all the double emulsion which had as a dispersed phase the o/w nanoemulsion 4E4T20 (4% wt Tween 20, 4% wt EVOO and 92% wt water) and those with 3% or 5% wt dispersed phase the nanoemulsion 6E6T40 (6% wt Tween 40, 6% wt EVOO and 88% wt water) showed bimodal distribution with high polydispersity(PDI>0.3). Moreover, the DSC thermograms for all double emulsions with dispersed phase the nanoemulsion8E8T20(8% wt Tween 20, 8% wt EVOO and 84% wt water) and those with high volume fraction (7% wt) of the dispersed phase 6E6T40 had undergone phase separation. In addition, in the current study, multiple o1/w/o2nanoemulsions fortified with CLA or CoQ10 were produced using extra virgin olive or olive pomace oil, and polyphenols from olive kernel were also incorporated in order to enhance their kinetic and chemical stability. All nanoemulsions showed bimodal droplet size distribution, and Newtonian behavior while polyphenols facilitated their homogenization. Droplet size distribution graphs showed bimodal behavior coming in agreement with the high PDI values. It was observed that the CLA-loaded o1/w/o2 nanoemulsions resulted to low MDD (393 ± 13 nm), also the incorporation of polyphenol compounds facilitated the homogenization process lowering further the MDD (335,7 ± 10,9 nm). The size distribution graphs obtained were similar for both vegetable oils, indicating that the differences in droplet size observed in the inner o/w nanoemulsion did not significantly affect the final droplets. The viscosity of the Newtonian multiple nanoemulsions ranged from 74.3 ± 2,4 to 94.3 ± 3,3 cP. The CoQ10-loaded nanoemulsions with OPO presented the lowest viscosity values (74.3 ± 2,4cP). Moreover, it was observed that nanoemulsions with higher viscosity also exhibit a higher MDD (499 ± 15 nm, 91,9 ± 1,3 cP), as their rheological behavior is heavily associated with the droplet size. The addition of polyphenolic compounds contributed to low viscosity values.As far as the kinetic and chemical stability of the sample is concerned, both vegetable oils resulted in samples with high bioactive retention values (>80%) and high ESI% values (>90%) after 30 days storage at 4 oC or 25 oC. Extra virgin olive oil resulted in more stable nanoemulsions in regards to kinetic and chemical stability at 4 oC, showing limited creaming and sedimentation boundary. The droplet size increased significantly during the storage period, with MDD growth being higher during refrigerated (increase > 260 nm) storage than at 25 oC (increase > 150 nm). The ESI% presented similar results to the MDD during storage. After 30 days, the MDD of all samples increased above 500 nm regardless the storage temperature. As far as the retention value is concerned, the concentration of CLA and CoQ10 was significantly decreased during storage. The addition of polyphenols, was proved beneficial lowering the retention value decrease. Moreover, the retention of the bioactive compounds was affected by the storage temperature with the lowest concentration of CLA and CoQ10 recorded at 25 oC after 30 days (R%CLA = 79.2 ± 0.90% and R%CoQ10 = 84,8 ± 4.22%). The reduction of TPC during storage was affected by the type of lipid phase and the incorporation of CLA or CoQ10, as the TPC of EVOO samples fortified with CoQ10 appeared to be more stable after 30 days of storage, regardless the storage temperature. However, the antioxidant activity of o1/w/o2 nanoemulsions was significantly affected by the storage period and temperature, as the DPPH values were more decreased at 25 oC compared to 4 oC after 30 days of storage. Additionally, it was observed that samples with CoQ10 and polyphenols exhibited the highest antioxidant activity even after 30 days. FTIR spectroscopy was used for the characterization of the interactions between the incorporated lipophilic compounds, the polyphenols and the lipid phase on molecular level before or after ultrasound homogenization. The incorporation of CLA or CoQ10 and the presence of polyphenols did not change the infrared spectrum of EVOO or OPO, indicating that no chemical interactions inside the emulsified system. Spectral changes occurred during aging, and led to a significant decrease of the CI (decrease of νC=O intensity at 1744 cm-1) for all samples. Also, FTIR spectra of multiple o1/w/o2nanoemulsions before and after ultrasound homogenization indicated that the sonication process had no significant effect on the composition of the lipid phase, especially in the case of EVOO (decrease of 6.75 ± 0.52%). A significant decrease of the CI was noted in the CLA-loaded o1/w/o2nanoemulsions (decrease of 24.10 ± 1.02%), while CoQ10 proved to protect the lipid phase by increasing the resistance to oxidation (decrease of 13,47 ± 0,67%). Moreover, the addition of polyphenolic compounds enhanced the stability of o1/w/o2nanoemulsion as well, especially in those containing OPO. Finally, the double structure of all the o1/w/o2 formulations was confirmed upon microscopic observation. Confocal scanning laser microscopy (CLSM) was used to visualize the microstructure changes depending on experimental conditions. All o1/w/o2 samples displayed similar structures. The images clearly demonstrated that both aqueous and lipid phases can be differentiated with a satisfying signal-to-noise ratio. The majority of emulsions did not formulate clusters, but they had a broad droplet size distribution. The majority of droplets had entrapped one small lipid droplet, however large spherical or irregular aqueous phases droplets with numerous oil droplets entrapped were also observed. Moreover, a large number of small oil droplets with a narrow size distribution were entrapped within the aqueous droplets. The double o1/w/o2emulsions with Span 20 were coarse and heterogeneous with many large accumulated droplets. Respectively with Tween 40, the structure became more homogeneous and the network was composed of finer spherical. Increasing the dispersed volume fraction, regardless the emulsifier type used, the presence of clusters was more frequent and the o1/w droplets were large. The multiple o1/w/o2 nanoemulsions with OPO were homogeneous and the network was composed of fine spherical droplets. However, EVOO multiple nanoemulsions were coarse and heterogeneous with many large accumulated droplets. Furthermore, the incorporation of CLA or CoQ10 had no significant effect on the structure of the o1/w/o2 multiple nanoemulsions, as no visible differences were observed using CLSM imaging. While the incorporation of polyphenols led to more spherical and homogenous o/w droplets implying that the presence of polyphenols can facilitate homogenization. Therefore, the study reveals that ultrasonic emulsification provides a simple, quick and effective method for the production of multiple o1/w/o2 nanoemulsions using low amounts of emulsifier. In addition, o/w and o1/w/o2nanoemulsions based on extra virgin olive or olive-pomace oil loaded with lipophilic compounds and phenolic extracts can be excellent delivery systems with satisfactory kinetic and chemical stability.
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