Kaktüs Meyvesinden (Opuntia ficus-indica L.) Betalainlerin Yeni Yöntemlerle Ekstraksiyonu, Niyozomal Enkapsülasyonu ve Gıda Uygulaması

Abstract

Prickly pear (Opuntia ficus-indica L.) is a fruit commonly cultivated in arid and semi-arid regions and is noteworthy rich in betalain pigments and various phenolic compounds. In recent years, betalains have grown in interest due to their visual and functional properties. Particularly, betalains are accepted as a natural alternative with rising consumer sensitivity to the synthetic colorants. However, the limited stability of these naturally derived compounds against environmental factors such as light, temperature, and pH could restrict their applicability. Therefore, the utilization of betalain-rich sources like cactus fruit and strategies to enhance the stability of these compounds in food systems has gained significance.In traditional extraction of natural pigments like betalains, the extended processing times, high solvent usage, and low extraction yield, are major limitations. Accordingly, there has been increasing interest in green extraction technologies, microwave and ultrasound-assisted methods, providing environmentally friendly, rapid, and high-yield alternatives. Furthermore, various encapsulation techniques have developed to enhance the stability of the extracted functional color pigments and other bioactives. For this purpose, niosomal encapsulation, a technology utilized for a long time in the pharmaceutical and cosmetic industries but relatively novel in food applications, has drawn attention as a promising carrier system for the delivery and controlled release of natural bioactives. Bioactive compounds such as betalains and phenolics not only have functional properties but also exhibit antimicrobial activity against certain pathogenic microorganisms. In addition, the application of these niosomal encapsulated bioactives to food systems contributes to both value-added product development and enhancement of bioaccessibility during digestion. Consequently, it is crucial to perform in-vitro digestion studies on extracts, niosomal capsules containing bioactive compounds, and niosome incorporated food matrices. To the best of our knowledge, there has been no investigation in the literature consisting of the extraction of betalains and phenolics from prickly pear using both ultrasound-assisted and vacuum microwave-assisted extraction methods in comparison with conventional method, also optimizing extraction conditions. Similarly, niosomal encapsulation of extracts, the optimization of encapsulation parameters, characterization of the niosomes, and application in a food matrix have not been previously reported. In this study, firstly extracts were prepared using conventional, ultrasound assisted, and vacuum microwave assisted extraction techniques, the extraction parameters were optimized, and extraction methods were compared. Since the promising results were obtained in vacuum microwave assisted extraction, the extract obtained in optimal extraction conditions was used in niosomal encapsulation process. During encapsulation, process parameters were optimized, the resulting niosomal formulation was characterized, and incorporated into a yogurt product. Finally, the effects of both the extract and the extract-loaded niosomes on the yogurt samples were analyzed, and in-vitro digestion studies were performed to evaluate the bioaccessibility of the bioactive compounds.In the beginning of the thesis, conventional extraction (CE), ultrasound assisted extraction (UAE), and vacuum microwave assisted extraction (VMAE) methods were used to extract betalains and phenolic compounds from prickly pear. Extraction conditions were optimized using a Box–Behnken design with 29 runs and four independent variables at three levels. For CE and UAE, variables were determined as extraction time (X₁) 1–3 hours and 30–90 min, extraction temperature (X₂) 30–50 °C, ethanol concentration in solvent (X₃) 40–80%, and sample-to-solvent ratio (X₄) 1:10–1:30 (g/mL). For VMDE, variables were vacuum pressure (X₁) 100–400 mmHg, extraction time (X₂) 2–8 min, ethanol concentration in solvent (X₃) 20–60%, and sample-to-solvent ratio (X₄) 1:10–1:30 g/mL. Total phenolic content (TPC), total antioxidant capacity (DPPH and CUPRAC methods), and total betalain content (TBC) were the responses. Second-order polynomial equations and variance analysis were obtained for each response using Response Surface Methodology. The optimization of extraction parameters was carried out for each extraction method. For CE, the optimum extraction conditions were an extraction time of 2.05 h, extraction temperature of 50 °C, 80% ethanol, sample-to-solvent ratio of 1:22.6 g/mL. For UDE the optimum extraction conditions were an extraction time of 30 min, extraction temperature of 50 °C, 40% ethanol, sample-to-solvent ratio of 1:30 g/mL. For VMDE the optimum extraction conditions were the vacuum value of 108.82 mmHg, extraction time of 2.47 min, 20% ethanol, sample-to-solvent ratio of 1:29.42 g/mL. Under optimal conditions, TPC values were 6.13, 6.38, and 7.79 mg GAE/g DW; TACDPPH were 13.96, 13.53, and 13.74 mmol TE/kg DW; TACCUPRAC were 34.87, 49.38, and 47.06 mmol TE/kg DW; and TBC values were 418.83, 471.82, and 715.50 mg betalain/kg DW for CE, UAE, and VMDE respectively. After optimization of the extraction methods, Q-TOF LC/MS analysis was performed to analyze betalain composition in extracts obtained in optimum conditions. The amounts of different types of betalains were calculated in terms of betanin standard equivalents. Antimicrobial activities of the extracts were investigated against Salmonella enteritidis subsp. enterica serovar Enteritidis (ATCC 13076), Escherichia coli O157:H7 (ATCC 25922), and Listeria monocytogenes via well diffusion and broth macrodilution methods. Extract obtained via VMDE exhibited activity against E. coli O157:H7 (ATCC 25922), while all extracts showed antimicrobial activity against Salmonella enteritidis subsp. enterica serovar Enteritidis (ATCC 13076). No inhibitory effect was detected against Listeria monocytogenes among extraction methods in well diffusion method. In brothmacrodilution method, the minimum inhibitory concentration (MIC) of extracts obtained in optimum extraction conditions was found only for Salmonella enteridis subsp. enterica serovar Enteritidis (ATCC 13076) in undiluted tubes. Since VMAE was founded as the best extraction method among other methods, niosomal encapsulation was performed using the extract obtained in optimum conditions of VMAE. Encapsulation conditions were optimized by response surface methodology with four independent variables at three levels: molar ratio of Span 60:Tween 80 molar (X₁) 1:1-3:1), the amount of the extract (X₂) 5–15 mg/100 mgsurfactant, the amount of the cholesterol (X₃) 0.1–0.3 mg/100 mg surfactant, and sonication time (X₄) 0–90 s. Responses were chosen as encapsulation efficiency (%), total phenolic content (TPC), total antioxidant capacity (TAC), b* value in color analysis, and total betalain content (TBC). Optimum encapsulation conditions were determined as molar ratio of Span 60:Tween 80 1.161, 14.44 mg extract/100 mg surfactant, 0.1 mg cholesterol/100 mg surfactant, and 89.99 s of sonication. In optimum conditions, the encapsulation efficiency, 96.06%; TPC, 3.06 mg GAE/g DW; TAC, 24.41 mmol TE/kg DW; b*, 2.81 and TBM, 687.30 mg betalain/kg DW. In niosomal capsule obtained in optimum encapsulation conditions, the betalain degradation kinetics over 30 days, evaluation of thermal stability, FT-IR, TEM, zeta potential, and particle size analyses were performed. In CE, UAE, and VMDE the thermal stability values of 55.5%, 60.8%, and 66.8% respectively increased to 92.91% after niosomal encapsulation. In the next stage of the thesis, for food application, yogurt was produced and optimal VMDE extract and optimum niosomal capsules were supplemented to yogurt at different concentrations (2-6%). Yogurts were stored at 4°C for 14 days, and the changes in physicochemical (pH, water holding capacity, dry matter, color, water activity), textural, rheological, microbiological (lactic acid bacteria count), and functional parameters (TPC, TAC and TBC) were analyzed, and results were compared with plain yogurt sample without any additives. Results indicated that bioactive properties were increased compared to plain yogurt leading to the development of a product with functional properties. Up to 4% concentration, yogurt quality maintained considerably in terms of physicochemical, textural and rheological characteristics. Niosome-added samples demonstrated better stability of bioactive compounds and betalains compared to extract-added samples during storage.In the final stage of the study, the simulation of in-vitro digestion was performed to evaluate the bioaccessibility of betalains and phenolic compounds in extract, extract loaded niosomes, and yogurt samples. After in-vitro digestion stages, TPC, TAC, and TBC were measured and compared with the results before digestion. Results showed that betalains and phenolic compounds declined in the gastric phase followed by an increase in the intestinal phase. In conclusion, in this study the potential of betalains and bioactive compounds extracted from prickly pear was investigated in terms of usage of natural food colorants and functional additives. Among the novel extraction technologies, vacuum microwave-assisted extraction (VMAE) was found to be the most efficient and environmentally friendly alternative compared to other extraction methods. Furthermore, it was determined that niosomal encapsulation is an effective technique for stabilization of betalains and sensitive bioactive compounds extracted via VMAE. The direct and encapsulated application of natural colorant components into plain yogurt yielded promising results in the development of an innovative product with functional properties. Finally, the bioavailability of the extract obtained under VMDE optimal conditions, extract-loaded niosomal vesicles, and functional yogurt samples were evaluated by in- vitro digestion experiments. The behavior of bioactive compounds and betalains during digestion processes was analyzed in detail. These findings demonstrated the applicability of betalains in the food industry and contributed to the food integration of innovative technologies. Accordingly, in further studies, the applicability of niosomal encapsulation of betalains and bioactive compounds extracted via VMAE to various food could be tested, thereby increasing the diversity of functional products.