Horozibiği (Amaranthus Caudatus L.) Çiçeklerinden Betalainler ve Fenolik Bileşiklerin Vakum Mikrodalga-Destekli Ekstraksiyonu ve Enkapsülasyonu

Abstract

Amaranthus caudatus L., with its pink-purple flowers rich in betalains, has gained attention in recent years as a potential natural food colorant due to its high phenolic content and strong antioxidant capacity. However, the limited stability of bioactive compounds of the natural sources limits their applications. Because extraction with traditional methods takes a long time and requires high amounts of solvent, vacuum microwave-assisted technology, which is a new generation green extraction method that can provide more efficient extraction in shorter times, has been developed. Encapsulation techniques have been introduced to enhance the stability of these natural pigments and health-promoting bioactive compounds. Among these, niosomes are vesicular carriers composed of surfactants that facilitate the delivery of bioactive compounds. According to the literature review and common knowledge, no study has been found that extracts betalains and phenolic compounds from amaranth flowers using vacuum microwave-assisted extraction and subsequently encapsulates them in niosomal vesicular structures for stability enhancement. Therefore, in this thesis, betalain and phenolic compound extraction from amaranth flowers was performed using both conventional and vacuum microwave-assisted extraction methods, and the results were comparatively analyzed. Subsequently, niosomal encapsulation was applied to improve the stability of the extracted pigments and bioactive compounds. The optimization of process conditions and formulations for extraction and encapsulation was conducted using the response surface methodology (RSM). Maceration was used as the conventional extraction (CE) method, while vacuum microwave-assisted extraction (VMAE) was selected as the new-generation extraction technique. A Box-Behnken design was applied with four independent variables at three levels for both methods. The independent variables for CE were extraction time (1, 2, and 3 hours), temperature (30, 40, and 50°C), ethanol concentration (20%, 40%, and 60%), and sample-to-solvent ratio (1:10, 1:20, and 1:30 g/mL). For VMAE, the independent variables were pressure (150, 300, and 450 mmHg), extraction time (3, 9, and 15 min), ethanol concentration (20%, 40%, and 60%), and sample-to-solvent ratio (1:10, 1:20, and 1:30 g/mL). The total betalain content (TBC), total phenolic content (TPC), and total antioxidant capacity (TACDPPH and TACCUPRAC) were analyzed for all extract samples. The response values obtained from CE experiments ranged between 1554.67–2918.67 mg betalain/kg dry weight for TBC, 9.19–14.81 mg GAE/g dry weight for TPC, 34.08 109.55 mmol TE/kg dry weight for TACDPPH, and 107.53–167.02 mmol TE/kg dry weight for TACCUPRAC. In the VMAE method, the response values ranged between 993.54 3153.58 mg betalain/kg dry weight for TBC, 5.54–17.04 mg GAE/g dry weight for TPC, 21.86–101.42 mmol TE/kg dry weight for TACDPPH, and 69.42–195.39 mmol TE/kg dry weight for TACCUPRAC. The optimization results revealed the optimal CE conditions as 1hour extraction time, 50°C temperature, 40.99% ethanol concentration, and a 1:10 g/mL sample-to-solvent ratio. For VMAE, the optimal conditions were determined as 450 mmHg pressure, 12-minute extraction time, 30.74% ethanol concentration, and a 1:23.91 g/mL sample-to-solvent ratio. The morphology of extracts obtained under optimal conditions was examined using scanning electron microscopy (SEM), revealing that CE resulted in a more fragmented and irregular cell structure, whereas VMAE produced smoother and more porous structures. Additionally, betalain composition analysis was performed using Q-TOF LC/MS, and the betalain content of extracts obtained under optimal conditions was calculated as 6393.17 ppm for CE and 9256.50 ppm for VMAE in betanin equivalents. The results demonstrated that VMAE achieved more efficient extraction than CE, reducing the processing time by 80% while using lower ethanol concentrations. The TBM value of untreated amaranth flowers was found to be 38.26% with KE and 44.27% with VMDE. Therefore, extracts obtained under the optimal VMAE conditions were selected for niosomal encapsulation. The response surface methodology was applied to optimize formulation parameters and ultrasonication time, with the independent variables being the molar ratio of surfactants (Span60:Tween80, mol:mol), extract-to-surfactant ratio (mg extract/100 mg surfactant), cholesterol-to-surfactant ratio (mg cholesterol/100 mg surfactant), and ultrasonication time (s). The experimental design followed a Box-Behnken model, and responses included total betalain content (TBC), total phenolic content (TPC), total antioxidant capacity (TACCUPRAC), and red color intensity measurements. The optimization results identified the optimal conditions as a 1:1 molar ratio of surfactants (Span60:Tween80), 8.109 mg extract/100 mg surfactant, 0.1 mg cholesterol/100 mg surfactant, and 81.877 sultrasonication time. Additionally, thermal stability, stability after 30 days of storage, TEM, FT-IR, particle size, and zeta potential analyses were conducted for the optimized niosome samples. According to the thermal stability experiments, the KE and VMDE thermal stability rates, which were found to be 67.35% and 65.73%, were increased to 97.974% with the niosome method. In conclusion, this study showed that the extraction of betalains and phenolic compounds from amaranth flowers can be efficiently performed using VMAE, which offers advantages over conventional methods in terms of time efficiency and solvent usage. Furthermore, niosomal encapsulation proved to be an effective strategy for enhancing the stability of betalains and phenolic compounds. In conclusion, VMAE was found to be a more sustainable and efficient technique than conventional extraction for obtaining betalains and phenolics from A. caudatus, with lower ethanol usage and shorter processing time. Moreover, niosomal encapsulation provided an effective strategy to improve the stability of these bioactive compounds, suggesting that the combined use of VMAE and niosomal encapsulation holds strong potential for the development of natural colorants for the food industry.