غنی‌سازی نوشیدنی کامبوجا با نانولیپوزوم‌های حاوی عصاره‌های برگ به و زیتون : ویژگی‌ها و اثرات

Introduction: Nanoliposomes, also known as nanoliposomal carriers, are spherical vesicles composed of phospholipid molecules arranged in a double-layered lipid bilayer. In this structure, the polar head groups face the aqueous exterior and interior, while the nonpolar (hydrophobic) tails orient inward, forming a hydrophobic core. This unique architecture enables nanoliposomes to encapsulate both hydrophilic and lipophilic compounds effectively (García-Morales et al. 2025). Nanoliposomes have proven successful in carrying a wide array of bioactive agents, including genetic material (e.g., DNA and peptides), proteins, vaccines, enzymes, and various drugs with anticancer, antimicrobial, antioxidant, antihemolytic, and anti-inflammatory properties (Carugo et al. 2016). Recent studies have increasingly explored the use of nanoliposomes in food systems to improve nutritional quality and functional benefits. Key factors influencing efficient encapsulation and controlled release include the encapsulation efficiency, process yield, sustained release profile, particle size, and zeta potential of the vesicles (Carugo et al. 2016).
Phospholipids, the fundamental components of liposomes, feature a polar (hydrophilic) head and nonpolar (hydrophobic) tails. When exposed to an aqueous environment, these molecules spontaneously assemble into spherical structures. The hydrophilic heads face the external and internal aqueous phases, creating an aqueous core that can encapsulate water-soluble compounds. Meanwhile, the hydrophobic tails align inward, forming a lipophilic bilayer capable of housing fat-soluble substances (D. Sharma, Ali, and Trivedi 2018). The chemical composition of the phospholipid head group, which typically includes choline, phosphate, and glycerol, determines the liposome’s surface charge, which can be neutral, positively charged (cationic), or negatively charged (anionic). The hydrophobic tails consist of one or two fatty acid chains, generally containing 14 to 18 carbon atoms. These chains may be saturated or unsaturated, influencing the membrane’s flexibility and permeability (Nsairat et al. 2022). The thin-film hydration method is the classical and most widely employed technique for producing liposomes and nanoliposomes. Initially, phospholipids are dissolved in volatile organic solvents like chloroform or methanol. Using a rotary evaporator, the solvent is carefully evaporated, leaving behind a thin lipid film coated onto the inner surface of the flask. This lipid film is then hydrated with an aqueous solution at a temperature above the lipid phase transition temperature, typically between 50 and 60℃, triggering spontaneous formation of multilamellar vesicles. These multilamellar structures can be further processed into small unilamellar vesicles through size-reduction techniques such as probe sonication or extrusion (Lombardo and Kiselev 2022).
In recent years, the food industry has increasingly focused on developing functional food products that deliver health benefits beyond basic nutrition. These foods contain bioactive compounds, either naturally occurring or introduced during processing, which can influence specific biochemical and physiological processes in the body when consumed. The incorporation of nanoliposome -enhanced ingredients into foods has generated considerable interest, as these nanocarriers may boost health by providing antioxidant, antihemolytic, antimicrobial, anti-inflammatory, photoprotective, and anticancer effects, along with controlled and sustained release of active compounds (Zarrabi et al. 2020). Advanced encapsulation technologies are now employed to protect the integrity and functionality of these bioactive food components and nutraceuticals, enhancing their stability and bioavailability. Nanocarriers offer numerous advantages to food manufacturers, including minimizing nutrient loss and enabling time-controlled delivery within food formulations (Vasconcelos et al. 2020).
Quince (Cydonia oblonga) and its various parts, including leaves, seeds, and fruits, are rich sources of polyphenols, organic acids, and flavonoids. These compounds contribute to the plant’s notable antioxidant, anti-inflammatory, blood sugar-lowering, and lipid-reducing effects. Similarly, olive leaf extract, derived from the leaves of Olea europaea, shows great potential as a nutraceutical for supporting cardiometabolic health. This extract is especially abundant in phenolic secoiridoids like oleuropein, hydroxytyrosol, and verbascoside, which are well-known for their powerful antioxidant, anti-inflammatory, and vascular-protective properties (Sardelli et al. 2025). Plant residues and biological waste have long been considered useless materials and environmental threats. Quince and olive leaves, due to their bioactive compounds, have been recognized as valuable plant materials in scientific research. These compounds possess strong antimicrobial and antioxidant properties, which can be effective in improving health and preventing the spoilage of food products. Encapsulation has been proposed as a promising approach to preserve bioactive food compounds and mask undesirable flavors. Nanoliposomes are one of the encapsulation carriers now widely used in the food and pharmaceutical industries (Kazan 2025; Tavakoli et al. 2018). Kazan (2025) employed microwave-assisted extraction (MAE) to obtain extracts from quince leaves (Cydonia oblonga). The extract obtained under optimal conditions was encapsulated using a solvent evaporation method. Nanoparticles containing the extract and blank nanoparticles were evaluated in terms of size, morphology, and thermal properties. For MAE, the optimal conditions were determined as 39.52℃, 300 W microwave power, and 15 minutes. Under these conditions, the total phenolic content was 218.18 mg.GAE/g, total flavonoid content was 63.8 mg.GAE/g, and the EC50 value was 9.45 μg/mL. The quince leaf extract was encapsulated in protein-based nanoparticles, with an average particle size of 89.9 nm and an encapsulation efficiency of 74.4%. Moreover, the thermal stability of the leaf extract was enhanced in its encapsulated form compared to its free form (Kazan 2025). In another study, Jami et al. (2024) investigated nanoliposomes containing anthocyanins from black fig and their application in kombucha beverages. They prepared nanoliposomes with different lecithin–cholesterol ratios (9:1, 8:2, 7:3, and 6:4) using the solvent injection method. They reported that the average particle size (hydrodynamic diameter) and particle size distribution for the different lecithin–cholesterol ratios ranged from 132 to 740 nm and 0.41 to 0.47, respectively. Additionally, the zeta potential values ranged from –26 to –42 mV. Ultimately, the samples with a 9:1 lecithin–cholesterol ratio were used in the formulation of the kombucha beverage. Sensory evaluation of the prepared beverages showed no significant differences among the samples in terms of aroma, mouthfeel, and overall acceptance (P > 0.05). The results indicated that nanoliposomes are an efficient system for the encapsulation of anthocyanins(Jami, Fathi-Achachlouei, and Shaddel 2024).
Kombucha’s popularity has surged recently, largely due to its reputation as a healthy fermented beverage. Its origins date back to approximately 220 B.C. in northeastern China during the Qin Dynasty, where it was known as the “Tea of Immortality” in Chinese folklore. This drink was highly valued for its health-promoting properties, which stem from its rich content of antioxidants, antimicrobial agents, and immune-enhancing compounds (Jayabalan and Waisundara 2019). These benefits arise not only from the tea or other substrates used but also from the metabolic activity of its symbiotic culture of bacteria and yeast. While the precise origins of kombucha remain somewhat debated, it is generally accepted that the drink spread from China across Asia and eventually into Europe via trade routes such as the Silk Road. During the COVID-19 pandemic, kombucha attracted renewed attention as a potential immune booster, with recognized antimicrobial and antiviral effects. The early 21st century marked a revival for kombucha, fueled by increasing consumer interest in functional foods and probiotic-rich fermented products (Andrade et al. 2025). In this study, the enrichment of quince leaf and olive leaf extracts in the form of nanoliposomes at different concentrations was investigated in kombucha beverage. The aim of this research was to examine the physicochemical, morphological, and microbial characteristics of nanoliposomes containing QLE and OLE, and to evaluate their application in kombucha, thereby taking a step toward the development of health-oriented fermented beverages.
Material and methods: Multilayer nanoliposomes encapsulating quince and olive leaf extracts were prepared using a modified thin-film hydration method.
Phosphatidylcholine and cholesterol at a mass ratio of 4:1 were dissolved in 99% ethanol. The solvent was evaporated under vacuum at 50℃ to form a thin lipid film. The film was then hydrated with deionized water containing the plant extracts at concentrations of 1000, 3000, and 6000 ppm. The resulting mixture was subsequently homogenized and sonicated to reduce the vesicle size. The characteristics of the nanoliposomes—including particle size, polydispersity index (PDI), and zeta potential—were measured by dynamic light scattering (DLS). The encapsulation efficiency (EE) was determined using UV-Vis spectrophotometry, and the physical stability was assessed over 60 days of storage at 25 °C. Fourier-transform infrared (FTIR) spectroscopy was performed to evaluate molecular interactions. The optimized nanoliposome formulations were incorporated into kombucha at four concentrations (100–400 ppm). The physicochemical properties (pH, °Brix), microbial counts, and sensory attributes of the enriched kombucha were then evaluated.
Results and discussion: The characterization of nanoliposomes loaded with QLE and OLE revealed distinct effects of extract type and concentration on particle size, PDI, and zeta potential. OLE-loaded nanoliposomes exhibited a significant increase in particle size from 173.4 nm to 312.77 nm as extract concentration increased from 1000 to 6000 ppm (P<0.05.), while QLE-loaded nanoliposomes showed an opposite trend, with particle size decreasing from 158.13 nm to 123.97 nm. PDI values for all samples were within an acceptable range (0.197–0.531), indicating relatively uniform particle distributions, particularly for QLE nanoliposomes. Zeta potential measurements confirmed good electrostatic stability for both extract types, remaining above the ±30 mV threshold, although higher OLE concentrations slightly reduced the negative charge. EE was high for all formulations (82–95%), reflecting effective loading of hydrophilic phenolic compounds into the aqueous core of the nanoliposomes. FTIR analysis confirmed physical interactions between the extracts and the lipid matrix without chemical bonding, indicating successful encapsulation within the phosphatidylcholine/cholesterol bilayers.
The application of the optimized nanoliposomes in kombucha demonstrated beneficial effects on physicochemical, microbial, and sensory properties. The enriched beverages showed increased pH (up to 4.01 for QLE3) and slightly reduced °Brix, with values remaining within safe consumption limits. Microbial analysis indicated a significant reduction in total viable counts and yeast/mold populations with increasing nanoliposome concentration, highlighting the antimicrobial potential of encapsulated phenolic compounds. Sensory evaluation revealed improved taste, odor, color, and overall acceptability, particularly in beverages containing QLE3 and OLE3 nanoliposomes, suggesting that encapsulation effectively masked the extracts bitterness and enhanced consumer perception. Overall, these findings demonstrate that QLE and OLE nanoliposomes can effectively improve the stability, bioactivity, and sensory quality of functional beverages such as kombucha, with extract type and concentration being key determinants of their performance.
Conclusion: The application of optimized QLE and OLE nanoliposomes preserved the physicochemical and microbial quality of kombucha within standard limits while significantly improving its sensory attributes and overall acceptability. These findings highlight that QLE and OLE-loaded nanoliposomes offer an innovative and effective strategy for enhancing the functional and health-promoting properties of fermented beverages.