Impactul estimat al rezultatelor obţinute 

În ceea ce priveşte rezultatele ştiinţifice, considerate ca indicatori de succes pentru proiect, au fost obţinute şi testate diferite sisteme de tip nanoemulsii îmbogăţite în zeaxantină sau esterul acesteia, utilizând patru tipuri de uleiuri vegetale (cocos, măsline, floarea soarelui şi in), diverşi emulgatori şi tehnici de obţinere. Nanoemulsiile obţinute s-au dovedit a fi stabile la păstrare în timp, în condiţii relevante pentru utilizarea lor în aplicaţii alimentare (pH 5-8, temperaturi 4 şi 20ºC şi concentraţii moderate de săruri). De asemenea, s-a demonstrat faptul că bioaccessibilitatea zeaxantinei este superioară din nanoemulsiile ulei-în-apă pe bază de alimente decât din alimentele propriu-zise, dar este influenţată de caracteristicile uleiului utilizat. Prin metode performante (RAMAN mapping, HPLC-PDA) s-a evidenţiat preluarea şi transportul celular al zeaxantinei, atât în celule intestinale cât şi în celule retiniene. Rezultatele cercetărilor au fost diseminate prin publicarea a 6 articole ISI (dintre care 2 Q1, 2 Q2), încă un articol Q1 în curs de publicare, 2 capitole de cărţi în edituri internaţionale prestigioase (Springer, Elsevier) şi 12 participări la conferinţe internaţionale. De asemenea, au fost susţinute două teze de doctorat, ambele pe tematica proiectului.

Rezultatul cel mai semnificativ al proiectului PN-III-P4-ID-PCE-2020-1172 cu titlul Nanostrategii pentru îmbunătăţirea stabilităţii şi bioaccessibilităţii zeaxantinei constă în stabilirea unei metode eficiente de obţinere de nanoemulsii stabile îmbogăţite în zeaxantină, atât cu compuşi puri, cât şi pe bază de alimente (cătină, goji, spirulina). Aceste nanoemulsii (în special cele obţinute cu ulei de măsline sau ulei de cocos MCT) pot fi utilizate ca ingrediente stabile în alimente sau suplimente alimentare, cu bioaccesibilitate crescută pentru zeaxantină, un pigment carotenoidic esenţial pentru prevenirea degenerescenţei maculare senile şi altor afecţiuni degenerative. Mai mult, în cazul cătinei, astfel de emulsii bogate în zeaxantină, protejate prin încapsulare în alginat, pot fi obţinute şi prin extracţie ecologică a carotenoidelor din subprodusele rezultate la procesarea fructelor, permiţând astfel valorificarea superioară a fructelor în conceptul de economie circulară.

The estimated impact of the results obtained

Regarding the scientific results, considered as indicators of success for the project, different nanoemulsion systems enriched in zeaxanthin or its ester were obtained and tested, using four types of vegetable oils (coconut, olive, sunflower and flax), different emulsifiers and production techniques. The obtained nanoemulsions proved to be stable when stored over time, under conditions relevant for their use in food applications (pH 5-8, temperatures 4 and 20ºC and moderate concentrations of salts). It has also been demonstrated that the bioaccessibility of zeaxanthin is superior from food-based oil-in-water nanoemulsions than from food itself, but it is influenced by the characteristics of the oil used. Through high-performance methods (RAMAN mapping, HPLC-PDA), the cellular uptake and transport of zeaxanthin was highlighted, both in intestinal cells and in retinal cells. The research results were disseminated through the publication of 6 ISI articles (of which 2 Q1, 2 Q2), another Q1 article in the process of publication, 2 book chapters in prestigious international publishing houses (Springer, Elsevier) and 12 participations in international conferences. Also, two doctoral theses were defended, both on the subject of the project.

The most significant result of the project PN-III-P4-ID-PCE-2020-1172 entitled Nanostrategies for improving the stability and bioaccessibility of zeaxanthin consists in establishing an efficient method for obtaining stable nanoemulsions enriched in zeaxanthin, both with pure compounds and food-based (sea buckthorn, goji, spirulina). These nanoemulsions (especially those obtained with olive oil or MCT coconut oil) can be used as stable ingredients in food or dietary supplements, with increased bioaccessibility for zeaxanthin, a carotenoid pigment essential for the prevention of age-related macular degeneration and other degenerative conditions. Moreover, in the case of sea buckthorn, such emulsions rich in zeaxanthin, protected by encapsulation in alginate, can also be obtained by ecological extraction of carotenoids from the by-products resulting from fruit processing, thus allowing the superior utilization of fruits in the circular economy concept.

Achieved Results

2021

• Zeaxanthin and zeaxanthin dipalmitate were obtained from Physalis alkekengi L. sepals by column and thin layer chromatography at a level of purity comparable to commercial standards and at lower costs;

• Five vegetable oils and one animal fat were characterized in terms of fatty acid and carotenoid composition by GC-MS and HPLC-PDA;

• Oil in water (O/W) nanoemulsions doped with carotenoids were obtained by probe ultrasonic emulsification, in the presence of emulsifier. The average hydrodynamic diameter was variable depending on the type of oil (chain length, unsaturation degree) but lower than 100 nm; the polydispersity index was less than 0.3 and the surface was negatively charged (zeta potential);

• The emulsions obtained with solid fats (palm oil and cream) had diameters (100-250 nm) and polydispersity indices much higher than those obtained with liquid oils;

• Nanoemulsions with zeaxanthin dipalmitate had a larger diameter than those with free zeaxanthin, regardless of the type of fat used

• Very good incorporation yield of carotenoids in nanoemulsions were found for liquid vegetal oils;

• Polymer-coated O/W nanoemulsions (cationic polylysine and anionic polyglutamic acid) were obtained and characterized by DLS and SEM.

2022

• Oil-in-water nanoemulsions were prepared, with zeaxanthin (Z) and zeaxanthin dipalmitate ZP (0.05 mg/ml), using four types of oils that gave good results (C-coconut MCT, M-olive, S-sunflower and L-in), in the presence of the emulsifier Tween 20, by the method of sonication in the sample.

• All emulsions were exposed to various pH values (3,4,5,6,7,8), temperatures (4, 20, 37, 50, 60 oC) and salt NaCl concentrations (0, 25, 50, 100 and 250 mM), over a time interval of up to 30 days. The stability of the emulsions was evaluated by measuring the diameter and distribution of the particles (DLS and by determining the zeta potential.

• Nanoemulsions had a good visual stability after 30 days, at room temperature and in the dark. They were stable at temperatures suitable for possible industrial applications (refrigerated foods, nutraceuticals), namely at 4, 20 oC, at salt concentrations below 50 mM and at moderate pH values (5-8), even at longer storage period (15-30 days).

• The bioaccessibility of free zeaxanthin was higher than that of zeaxanthin dipalmitate, regardless the oil used

• The bioaccessibility of both carotenoids was higher for nanoemulsions compared to non-emulsified oils doped with carotenoids. Nanoemulsions with zeaxanthin obtained with coconut oil (over 60%) and those with zeaxanthin dipalmitate and olive oil (over 40%) showed the highest bioaccessibility, far superior to the bioaccessibility of zeaxanthin from known food matrices.

• The obtained nanoemulsions did not show cytotoxicity on intestinal and retinal cells for physiological concentrations (1 µM zeaxanthin). Cellular uptake and secretion of zeaxanthin in intestinal and retinal cells was superior from nanoemulsions compared to solvent delivery, and dependent on the type of emulsions. The highest values were obtained for nanoemulsions with unsaturated oils.

• The presence of zeaxanthin in cell cultures was qualitatively confirmed by Raman mapping and measures by C30-HPLC-PDA.

2023

• Three food matrices – sea buckthorn berries, goji berries and spirulina (powder) - were characterized in terms of carotenoids (HPLC-PDA) and fatty acids (GC-MS) composition

• Goji berries and sea buckthorn berries contain zeaxanthin mainly in esterified form, while in spirulina zeaxanthin is found only in free (unesterified form) (24.55 mg/100 g). Goji fruits have the highest content of total carotenoids (122.45 mg/100 g), in which zeaxanthin dipalmitate (105 mg/100 g) is the major compound.

• Sea buckthorn has the highest lipid content, with a high percentage of MUFA and SFA, while SFA (61%) and PUFA (33%) predominate in spirulina. Goji is rich in PUFA, with 56% linoleic acid.

• In vitro digestion (amended INFOGEST protocol) of matrices showed a better bioaccessibility of free zeaxanthin from sea buckthorn (42.3 %) and spirulina (38.1 %) compared to goji (7.3 %). The bioaccessibility of zeaxanthin was superior to the bioaccessibility of total carotenoids in sea buckthorn and spirulina samples.

• Unsaponified and saponified carotenoid extracts did not show cytotoxicity in Caco-2 cell culture at concentrations of 1 and 5 µM total carotenoids. Experiments carried out in transwells showed an uptake of free zeaxanthin between 1-8.5%, with the highest value for spirulina and very low values for goji. The transport and efflux of carotenoids in the basolateral medium ranged between 5.1-11.8 %, in a similar order to that of cellular uptake.

• Oil-in-water nanoemulsions (1:10) were prepared starting from the three food matrices rich in zeaxanthin using four types of oils (C-coconut MCT, M-olive, S-sunflower and L-flaxseed), and Quillaja saponin as surfactant (0.5 %), by probe sonication. The obtained nanoemulsions had mean diameters below 50 nm, polydispersity indices below 0.4, and were negatively charged (ζ-potential from -8.86 to – 30 mV).

• Starting from sea buckthorn oil emulsion, sodium alginate microspheres (700 µm) were also fabricated, with good encapsulation efficiency and stable at acidic pH.

• The stability of carotenoids in the obtained nanoemulsions was determined during storage for 30 days, at pH 3, 7 and 8, in the dark and at ambient temperature. The retention of carotenoids at neutral and slightly alkaline pH ranged between 62-90.5 %, while at acidic pH the stability was very low (7.2–10.2 %). The stability of carotenoids, at all pHs, was influenced by both the matrix and the oil used for emulsification. The best stability was recorded for nanoemulsions based on goji, coconut oil and olive oil, followed by sea buckthorn with olive oil and coconut oil.

2024

• Nanoemulsions were obtained from three food matrices – sea buckthorn berries, goji berries and spirulina (powder), using vegetable oils (coconut oil, olive oil, sunflower oil and linseed oil).

• The bioaccessibility of Zea, as determined by simulated in vitro digestion (INFOGEST protocol), is highly variable depending on the matrix, but significantly higher from food-based nanoemulsions compared to the corresponding food matrices.

• The highest bioaccessibilities were obtained for nanoemulsions with coconut oil (short/medium chain saturated fatty acids), respectively with olive oil (long chain monounsaturated fatty acids), regardless the food matrix.

• The cellular uptake and transport of Zea from food matrices varied in the order spirulina > sea buckthorn > goji, with higher values ​​for the emulsified forms, especially for spirulina and polyunsaturated oils.

• The RAMAN mapping technique and HPLC-PDA analyzes demonstrated the internalization of Zea in intestinal and retinal cells.