Παρασκευή σύνθετων νανογαλακτωμάτων τροφίμων και διατήρησή τους σε ψύξη

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 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.