Electrospinning Encapsulation of Chlorophylls Microwave-Extracted from Spinach Waste and Utilization as Colouring Agent in Yoghurt

Electrospinning Encapsulation of Chlorophylls Microwave-Extracted from Spinach Waste and Utilization as Colouring Agent in Yoghurt | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Electrospinning Encapsulation of Chlorophylls Microwave-Extracted from Spinach Waste and Utilization as Colouring Agent in Yoghurt Beyza Sukran Isik, Sinan Bayram, Filiz Altay This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5188303/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Natural colorants have become a developing market because of consumer preferences, and more cost-effective and stable production is necessary. In the present study, a natural green colorant was obtained via microwave-assisted extraction from spinach waste to produce affordable, environmentally-friendly and innovative colorants. The extracts were encapsulated with zein via uniaxial and coaxial electrospinning techniques to overcome the stability issues associated with their natural green color. The encapsulation efficiencies of uniaxially encapsulated chlorophyll extracted from spinach waste by microwave and coaxially encapsulated chlorophyll extracted from spinach waste by microwave were 63.50 and 49.53% for chlorophyll a and 57.18 and 88.58% for chlorophyll b , respectively. The microwave-assisted extraction and addition of eumelanin significantly improved the antioxidant activity and thermal stability of chlorophylls. The stability test against light revealed that 61% of chlorophyll a could be preserved by uniaxial electrospinning. In addition, when chlorophyll-loaded nanofibers were incorporated into yogurt, a more stable green color was achieved during storage. chlorophylls uniaxial electrospinning coaxial electrospinning electrospinning encapsulation spinach waste microwave extraction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. INTRODUCTION Spinach is a green leafy vegetable and a good source of chlorophyll pigments. While there are more than 100 different chlorophyll structures in nature, chlorophyll a and b are the most abundant in spinach (Murcia et al., 2020 ; Scheer, 2006). However, spinach is a highly perishable vegetable, and between 13% and 25% of its waste is produced from damaged leaves and solid residues during the juice production process (Derrien et al., 2017 ). Valuable bioactive compounds are still present in wastes, which can make spinach waste a considerable raw material for natural green colorants. The extraction of bioactive compounds from waste via novel methods has become popular especially microwave extraction which is considered a sustainable and green technique, providing an efficient solution for industrial applications (Dai & Mumper, 2010 ). The application of microwaves results in the rupture of plant cell walls, thereby decreasing the resistance to extraction by enhancing internal diffusion processes (Braga et al., 2022 ). Chlorophylls are lipid-soluble green pigments present in the plastids of a wide range of plants, algae, and certain bacterial species. There are numerous health advantages of chlorophylls. Chlorophyll has strong antioxidant characteristics and helps prevent cancer, diabetes, cataracts, and heart disease (Murcia et al., 2020 ). In recent years, a growing body of scientific literature has demonstrated the significant health benefits associated with chlorophyll consumption, including antimutagenic, and antigenotoxic effects. Moreover, various chlorophyll derivatives have been extensively investigated for their potential in preventing and treating specific conditions, such as cancer, infections, and dermatological disorders (Perez-Galvez et al., 2017 ). Owing to their vivid green color, chlorophylls also serve as food colorants and are safely incorporated into various products either in pure form or in complex with copper, such as food, cosmetics and oral hygiene products. However, some countries limit the use of copper complexes of chlorophylls in drugs and foods because of concerns regarding the potential toxicity of copper (Simpson et al., 2012 ). There has been a growing interest in natural food colorants, including chlorophylls. The global market for natural food color was valued at an estimated USD 1.33 billion in 2022. Projections indicate that this market will expand at a compound annual growth rate (CAGR) of 8.3% from 2023 to 2030 (Natural Food Color Market Size, Share & Trends Analysis, 2023). However, the instability of natural chlorophyll pigments due to exposure to light, heat, air, and extreme pH conditions limits their use in commercial areas (Hsiao et al., 2020 ). This problem can be overcome with encapsulation. There are many studies on the encapsulation of chlorophylls by spray drying (Kang et al., 2019 ; Ledri et al., 2024 ; Rajabi et al., 2024 ; Zhang et al., 2019 , 2020 ), freeze drying (Dewi et al., 2022 ; Ledri et al., 2024 ; Rajabi et al., 2024 ), fluid bed technology (Yang et al., 2022 ), liposomes (Chu et al., 2014 ), complex coacervation (Agarry, Wang, Cai, Kan, et al., 2022), droplet microfluidics (Hsiao et al., 2020 ), and ionic gelation (Tekin & Ersus, 2021 ). However, there is currently no documentation of the encapsulation of natural chlorophyll extracts by electrospinning techniques. Electrospinning is a simple, versatile top-down process that is promising because of its high encapsulation efficiency; improved bioavailability, light, thermal and storage stability; and advanced protection against chemical degradation (Anu Bhushani & Anandharamakrishnan, 2014 ; Rostamabadi et al., 2020 ). In this study, zein was selected as a coating material for electrospinning process. Zein is a type of prolamin storage protein that is typically found within protein bodies located in the endosperm of maize. Zein is recognized as an exceptional material for the electrospinning owing to its biodegradability, flexibility, nontoxicity, and fiber-forming characteristics (Wang et al., 2019 ). It was also selected specifically because of its thermal and water stability and high oxygen barrier properties (Deng et al., 2018 ; Wang et al., 2019 ). To improve chlorophyll sensitivity, this study aimed to utilize the melanin pigment in addition to zein. Melanins, heterogeneous polymeric pigments, show high levels of antioxidant activity because of their phenolic and indolic components and contribute to the removal of free radicals through reduction reactions (Altındağ et al., 2022 ; Kumar et al., 2024 ). They can absorb low-wavelength, high-energy rays such as gamma rays, X-rays and UV rays and may provide increased photostability to light-sensitive products. In addition, research emphasizes that there is an increase in the thermal stability of polymer products provided by the addition of melanin (Di Mauro et al., 2019 ; Shanmuganathan et al., 2011 ). In this study, the aim was to extract natural green colorants from spinach waste via different extraction techniques and improve their stability via uniaxial and coaxial electrospinning. The morphology, physicochemistry and stability of the fabricated nanofibers were further investigated, and these materials were subsequently added to yogurt as a coloring agent. 2. MATERIALS AND METHODS 2.1. Materials Spinach was purchased from a local market in Istanbul (Turkiye). Spinach leaves were washed, dried and kept at room temperature. After 4 days, spinach was no longer suitable for consumption and was considered spinach waste. Ethanol (purity of 99.9%) was purchased from Isolab. Liquid chromatography (LC)-grade methanol, acetone and dimethyl sulfoxide (DMSO) were acquired from Merck. Chlorophyll a , chlorophyll b and zein were procured from Sigma Aldrich. Alkali salts of copper chlorophyllins (E141 (ii)) were purchased from Alfasol Turkiye. 2.2. Methods 2.2.1. Extraction Fresh spinach and spinach waste were extracted via two different extraction methods which were traditional and microwave extraction. For traditional extraction, chlorophyll was extracted from fresh spinach and spinach waste via a previously described method with some modifications (Zhang et al., 2017 ). One hundred grams of spinach was milled with a grinder for 15 s using deionized water at a ratio of 1:2 (w/v) to remove water-soluble compounds. The resulting precipitate was then extracted with 200 ml of ethanol at 60°C for 30 min in a water bath. The solid and liquid parts were separated. The supernatant was transferred to a beaker, and the precipitate was re-extracted. The extraction mixture was placed in a refrigerator at 4°C overnight and centrifuged at 4000 rpm for 10 min at 4°C to separate the proteins. The supernatant was concentrated with a rotary evaporator at 40°C. Ethanol was added to the obtained solid extract at 0.05 g/mL and the extract was stored at -20°C until further analysis. The chlorophyll extracts extracted from fresh spinach and spinach waste via traditional method were coded as FTE and WTE, respectively. Microwave extraction was performed with a microwave oven (Samsung GE83XIAND). Spinach waste and raw spinach were homogenized with a grinder for 15 s with deionized water at a ratio of 1:2 (w/v) to remove water-soluble compounds, and the precipitate was collected as in the traditional method. One hundred grams of spinach was extracted in 1000 ml of ethanol. 100 W was applied for 10 min in the microwave oven. After application, the temperature was measured as 55°C. The solid and liquid parts were separated. The supernatant was incubated at 4°C overnight, centrifuged at 4000 rpm for 10 min at 4°C, and concentrated with a rotary evaporator, similar to the traditional method. Ethanol was added to the obtained solid extract at 0.05 mg/mL, and the mixture was stored at -20 o C until further analysis. The chlorophyll extracts extracted from fresh spinach and spinach waste via the microwave method were coded FMWE and WMWE, respectively. 2.2.2. Production of Eumelanin (Eu) Pigment The Eu pigment was produced by the Streptomyces parvus BSB49 strain. The production, polymerization and purification of the Eu pigment were performed according to the procedure outlined by Bayram et al ( 2020 ). 2.2.3. Uniaxial and coaxial electrospinning encapsulation The electrospinning was conducted with electrospinning equipment (Inovenso NE100, Turkiye) at room temperature. The equipment included a syringe pump (New Era Pump Systems Inc., NE-300, USA), tip of the needle, collector plate and high-voltage power supply (Nanofen, Ankara, Turkiye). The compositions and preparation conditions of the feed solutions are given in Table 1 . Zein was dissolved in 80% ethanol at 400 rpm for 2 h at room temperature. Chlorophyll extracts were mixed with zein solution, and the concentration of zein in the final solution was 30% (w/v) for uniaxial electrospinning. Zein solution was prepared at 30% (w/v) for coaxial electrospinning. The Eu solution was prepared with DMSO at 1 mg/mL. The shell and core materials were mixed before electrospinning via uniaxial electrospinning. The flow rates of the zein solution and chlorophyll extracts are listed in Table 1 . The nanofibers were fabricated on parchment paper, which was used for covering the collector plate. Table 1 Composition of the feed solution, electrospinning conditions and characteristics of the feed solutions Sample number Feed solutions Electrospinning conditions Feed solution characteristics* Core composition (A) Shell composition (B) Polymer solution (w/v) B1 Chlorophyll extract (v/v) B2 Mixing ratio (v/v) B1:B2 Electrospinning type Flow rate (mL/h) Applied voltage (kV) Distance to the collector plate (cm) Electrical conductivity (mS/cm) Surface tension (mN/m) Apparent viscosity (mPa.s) at 10 s − 1 1 - 30% zein (6 g) 80% ethanol (20 mL) - - Uniaxial 0.8 17 15 0.90±0.07 c 25.37±0.05 a 0.12±0.01 c 2 - 40% zein (6 g) 80% ethanol (15 mL) FTE (5 mL) 3:1 Uniaxial 0.8 17 15 0.81±0.02 b 25.20±0.56 a 0.14±0.01 d 3 - 40% zein (6 g) 80% ethanol (15 mL) WTE (5 mL) 3:1 Uniaxial 0.8 17 15 0.82±0.04 b 25.45±0.53 a 0.14±0.01 d 4 - 40% zein (6 g) 80% ethanol (15 mL) FMWE (5 mL) 3:1 Uniaxial 0.8 17 15 0.83±0.01 b 25.36±0.61 a 0.15±0.01 d 5 - 40% zein (6 g) 80% ethanol (15 mL) WMWE (5 mL) 3:1 Uniaxial 0.8 17 15 0.79±0.03 b 24.71±0.13 a 0.15±0.01 d 6 - 20% Zein (6 g) 80% ethanol (30 mL) + Eu (5 mg) DMSO (5 mL) WMWE (5 mL) 7:1 Uniaxial 1.5 25 10 0.73±0.01 a 29.26±0.07 b 0.09±0.01 b 7 - 25% zein (6.25 g) 80% ethanol (25 mL) + Eu (17.5 mg) DMSO (17.5 mL) WMWE (5 mL) 8.5:1 Uniaxial 1.5 25 10 0.72±0.01 a 29.13±0.01 b 0.05±0.01 a 8 FTE 30% zein (6 g) 80% ethanol (20 mL) - - Coaxial A: 0.3 B: 0.6 17 13 - - - 9 WTE 30% zein (6 g) 80% ethanol (20 mL) - - Coaxial A: 0.3 B: 0.6 17 13 - - - 10 FMWE 30% zein (6 g) 80% ethanol (20 mL) - - Coaxial A: 0.3 B: 0.6 17 13 - - - 11 WMWE 30% zein (6 g) 80% ethanol (20 mL) - - Coaxial A: 0.3 B: 0.6 17 13 - - - Eu: Eumelanin, FTE: chlorophyll extracted from fresh spinach via traditional extraction, WTE: chlorophyll extracted from spinach waste via traditional extraction, FMWE: chlorophyll extracted from fresh spinach via microwave extraction, WMWE: chlorophyll extracted from spinach waste via microwave extraction. *Data are presented as the mean ± standard deviation of three replicates. The values in each column with different letters indicate statistically significant differences ( p < 0.05). 2.2.4. Determination of Solution Properties To assess the electrospinnability of the feed solutions, electrical conductivity, surface tension and viscosity were measured in triplicate. The electrical conductivity of the feed solutions was determined via a conductometer (WTW LF95, Germany) at room temperature in triplicate for each solution. The surface tension of the feed solutions was measured by a tensiometer (Dataphysics DCAT 11 E, Germany) at room temperature in triplicate for each solution. The viscosity of each solution was measured with a rheometer (Haake Rheostress 1, Germany) via a plate-plate sensor (D = 35 mm, gap = 1 mm) at room temperature in triplicate for each solution. The shear rate was between 0.1 and 100 s − 1 . The results were modeled via software (Haake RheoWin3 Data Manager, Germany) according to the power-law equation: \(\:\tau\:=K{\dot{\gamma\:}}^{n}\) Eq. 1 where τ is the shear stress (Pa), K is the consistency index (Pa.s n ), n is the flow behavior index and \(\:\dot{\gamma\:}\) is the shear rate (s − 1 ). The apparent viscosity (η) of the samples was calculated at 100 s − 1 via the following equation: \(\:\eta\:=\text{K}{\dot{\gamma\:}}^{n-1}\) Eq. 2 2.2.5. Characterization of nanofibers The zeta potential of each sample was measured in triplicate via a dynamic light scattering instrument (Malvern Zetasizer Nano ZS, Worcestershire, UK) at room temperature. Distilled water was used as a dispersant. The nanofibers were dispersed into 0.2% (w/v) distilled water. The morphologies of the electrospun samples were determined by scanning electron microscope (SEM) (Zeiss Evo LS10, Germany) at high vacuum with an accelerating voltage. Electrospun samples measuring 1 cm 2 were coated with Au-Pd via a sputter coater (Quorum, SC7620). Images were taken at certain magnifications. The mean diameter of the fibers was determined via SEM images via ImageJ software with the DiameterJ plugin (National Institutes of Health, USA). The size distribution was evaluated via OriginLab software. The infrared spectra of the samples were obtained via an FTIR spectrometer (Jasco-4000, United Kingdom) with an attenuated total reflection (ATR) unit attached. The spectra of each sample were measured with 64 scans and a resolution of 4 cm − 1 . Scans were conducted over a spectral range of 400–4000 cm − 1 . The thermal properties of the samples were assessed via differential scanning calorimetry (DSC) (Q10, New Castle, USA) in triplicate for each sample. The spinach extracts, zein and synthetic colorant (E141 (ii)) (5 ± 0.1 mg) and nanofibers (3 ± 0.1 mg) were weighed in a DSC aluminum pan. An empty DSC-pan was used as an inert reference. The sample and the reference pans were then placed inside the calorimeter, cooled to 20°C at a speed of 5°C·min − 1 , and heated to 300°C at a heating speed of 10°C·min − 1 . 2.3. HPLC analysis The chlorophyll amount was determined via reversed phase high performance liquid chromatography (HPLC). Analysis was performed via an Agilent 1260 Infinity HPLC system (USA) coupled to a DAD detector (Agilent, G131D, USA). The compounds were separated via an ACE 5 C18 column (150 × 4.6 mm), and the column temperature was maintained at room temperature. The injection volume was 10 µL, and the samples were injected with an autosampler (Agilent 1329B, USA). An isocratic method was used, and the mobile phase was water:methanol:acetone (4:36:60, v/v/v). The flow rate was 0.6 ml/min. The eluted peaks were detected at 661 nm (Li et al., 2014 ). Chlorophyll standards and extracts were diluted with methanol/dichloromethane (65/35; v/v) (Gleize et al., 2012 ). The electrospun samples were first dissolved in methanol/dichloromethane (65/35; v/v), and then the zein was removed with water and acetone before HPLC analysis. 2.4. Antioxidant activity The antioxidant activity of the extracts and nanofibers was determined via 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity (DPPH) analysis (Braca et al., 2001 ). A total of 25 mg of nanofibers was dissolved in methanol. 100 µL extracts or nanofiber solutions were mixed with 3 mL of a 0.004% methanol solution of DPPH. After 30 min in the dark, the absorbance was read at 517 nm against methanol (R 2 = 0.96). 2.5. Light stability of chlorophylls The extracts and nanofibers were exposed to direct sunlight to determine the stability of the chlorophyll. First, 0.1 g of nanofibers was dissolved in 2 mL of ethanol (80%, v/v). Samples were collected at days 0, 1, 4, 7 and 10. The chlorophyll content was determined according to the aforementioned method. 2.6. The addition of chlorophylls to yogurt The chlorophyll-loaded electrospun samples (0.1 g) were mixed with yogurt. 5 ml of chlorophyll extracts and synthetic colorant (E141 (ii)) were added into yogurt and homogeneous end products were obtained. A colorimeter (Konica Minolta Chromameter CR-400, Japan) was used to determine the CIE L*, a*, b* values of the samples, where L* is the lightness of color (100 = white, 0 = black), a* value (+ a* = red, -a* = green), and b* value (+ b* = yellow, -b⁄*= blue). The measurements were conducted at days 0, 1, 3, 5 and 7. The total color difference (△E*) was determined via Eq. 3, as outlined in a previous study (Mercali et al., 2014 ). The formula is as follows: \(\:\varDelta\:{E}^{*}\:=\:\sqrt{{\left(\varDelta\:{a}^{*}\right)}^{2}\:+\:{\left(\varDelta\:{b}^{*}\right)}^{2}\:+{\left(\varDelta\:{L}^{*}\right)}^{2}}\) Eq. 3 where △E* represents the total color difference, △a* represents the difference between the a* values of the control and the sample, △b* represents the difference in b* values between the control and the sample, and △L* represents the difference in L* values between the control and sample. The synthetic colorant (E141 (ii)) was selected as a control sample. 2.7. Statistical analysis IBM SPSS Statistics 28 (Chicago, IL, USA) software was used for the statistical analysis. The data are presented as the mean ± standard deviation (n = 3). One-way analysis of variance (ANOVA) was conducted via Duncan’s multiple range test. Differences between means were considered significant at p < 0.05. 3. RESULTS AND DISCUSSION 3.1. Effect of the Extraction Method on the Chlorophyll Content The chlorophyll a and b contents of fresh and waste spinach extracted via traditional and microwave extraction are shown in Table 2 . According to the results, fresh samples contained higher chlorophyll a and b contents than waste samples. While the highest chlorophyll a content (13.62 ± 0.10 mg/g dry weight (DW)) was measured for fresh spinach extracted via the traditional extraction method (FTE), fresh spinach extracted via microwave extraction (FMWE) presented the highest chlorophyll b content (6.98 ± 0.22 mg/g DW). Table 2 Chlorophyll content of the extracts Sample Chlorophyll a (mg/g DW) Chlorophyll b (mg/g DW) FTE 13.62 ± 0.10 d 5.76 ± 0.11 c WTE 9.87 ± 0.19 b 3.89 ± 0.01 a FMWE 11.52 ± 0.14 c 6.98 ± 0.22 d WMWE 5.74 ± 0.01 a 4.63 ± 0.11 b FTE: Fresh spinach extracted via traditional extraction, WTE: Spinach waste extracted via traditional extraction, FMWE: Fresh spinach extracted via microwave extraction, WMWE: Spinach waste extracted via microwave extraction The data are displayed as the means ± standard deviations of three replicates. The values in each column with different letters are statistically significant (p < 0.05). The chlorophyll content of fresh spinach reported in the literature significantly varies. While one of the studies reported chlorophyll a and b contents in spinach at 37.4 ± 3.4 and 13.9 ± 2.0 mg/100 g, respectively (Dermesonluoglu et al., 2015 ), another study reported that 100 g of spinach contained 103.37 ± 3.94 mg of chlorophyll a and 32.89 ± 0.76 mg of chlorophyll b (Lee et al., 2005 ). Another study revealed 71 different spinach genotypes and reported that the chlorophyll a and b contents ranged from 475.76 to 1196.45 and from 200.91 to 522.60 nmol/g FW, respectively, in fall-grown spinach. The chlorophyll content of spinach was reported to range between 13.61 and 15.82 mg/g for chlorophyll a and between 9.16 and 10.18 mg/g for chlorophyll b in a recent study (Nipa et al., 2023 ). In spring-grown spinach, the chlorophyll a content varies from 646.66 to 1400.00 nmol/g, and the chlorophyll b content fluctuates from 219.26 to 474.46 nmol/g (Hayes et al., 2020 ). The phytochemical composition of spinach is affected by climatic conditions during the growing season (de Azevedo-Meleiro & Rodriguez-Amaya, 2005 ), nitrogen status (Rorie et al., 2011 ), leaf maturity (Farnham et al., 2012 ), cultivation methods (Koh et al., 2012 ), postharvest processing conditions (Leong & Oey, 2012 ; Martínez et al., 2013 ; Mendelová et al., 2014 ), and plant genotypes (Hayes et al., 2020 ; Kidmose et al., 2001 ). In addition, different extraction conditions and determination methods can lead to different results. Therefore, the chlorophyll content of the spinach extracts determined in this study may differ from the results reported in the literature. The chlorophyll contents of the waste samples were lower than those of the fresh samples. Chlorophyll degrades when exposed to light, air and heat, resulting in the loss of phytol side chain or the removal of central Mg 2+ (Simpson et al., 2012 ). The lowest chlorophyll a content (5.74 ± 0.01 mg/g DW) was detected in spinach waste extracted via microwave extraction (WMWE) and spinach waste extracted via traditional extraction (WTE) presented the lowest chlorophyll b content (3.89 ± 0.01 mg/g DW). In this study, spinach was kept at room temperature for 4 days, after which it was considered spinach waste. There are several studies concerning the chlorophyll content of spinach during storage. One of the studies measured the chlorophyll a and b contents of spinach stored at 5°C and 20°C. The chlorophyll a content decreased from 103. 37 ± 3.94 mg/100 g to 24.53 ± 4.84 mg/100 g at 20°C. Even though the chlorophyll b content increased from 32.89 ± 0.76 mg/100 g to 33.47 ± 2.02 mg/100 g, the content was 42.02 ± 2.26 mg/100 g on the first day of storage at 20 °C. The chlorophyll b content first increased during storage but then started to decrease on day 2 (Lee et al., 2005 ). Another study reported that the chlorophyll a content decreased continually to ≈65% of the initial level by day 4 at 25°C. Moreover, the chlorophyll b content decreased similarly to that of chlorophyll a but at a lower rate (Yamauchi & Watada, 1991 ). Similar results were also observed in this study. Although the rates of decrease in chlorophyll a and b changed, a decrease was recorded for both extraction methods. According to the results, the traditional extraction method resulted in a higher chlorophyll a content than did microwave extraction. These results can be explained in several ways. The most likely explanation could be the conversion of chlorophyll a to chlorophyll b because the chlorophyll b contents of the microwave samples were higher than those of the samples extracted via traditional method. The transformation of chlorophylls a and b , which is also called the chlorophyll cycle, is believed to yield either chlorophyll a or b , according to particular physiological needs (Rüdiger, 2002 ). The metabolism of chlorophyll b plays a crucial role in the mechanisms of light acclimation in plants (Hu et al., 2021 ). Chlorophyll a is crucial for photochemical processes, whereas chlorophyll b enhances a plant's ability to capture a broader spectrum of light, particularly because it has strong absorption at approximately 450 nm, a wavelength that chlorophyll a does not absorb efficiently. This characteristic makes chlorophyll b particularly important for increasing a plant's light-harvesting ability (Larkum et al., 2018 ). Furthermore, the biosynthesis and degradation of chlorophyll b are closely associated with the assembly and disassembly of light-harvesting complexes, which can be regulated according to varying light conditions (Tanaka & Tanaka, 2011 ). Vegetables continue to undergo biochemical processes due to their modular structure and ability to maintain physiological autonomy once harvested and placed on supermarket shelves. One of the studies investigated whether endogenous rhythms persist in commercial vegetables and, if so, how these cycles might influence their nutritional quality. It was observed that Chl a/b ratios oscillated under both constant light and darkness in packaged rocket leaves, whereas oscillations in total Chl a + b were detected exclusively under darkness conditions (De Larrinaga et al., 2019 ). The conversion could also be triggered during microwave extraction. The chlorophyll structure consists of four pyrrole groups linked by a central magnesium ion to form a tetrapyrrole (or porphyrin) ring system, with a 20-carbon hydrocarbon side chain known as a phytol group. The central magnesium atom of chlorophyll may be sensitive to microwaves as a result of the ionic conduction mechanism (Yan et al., 2023 ). Interactions between the central magnesium atom and microwaves may cause localized heating or changes in the electronic structure around the magnesium atom, potentially affecting the stability of the chlorophyll molecule. The chlorophyll binding site is situated in a predominantly hydrophobic environment, with an estimated dielectric constant (ε) of approximately 2, which is representative of the typical optical dielectric constant observed in organic molecules (Krishtalik et al., 1997 ). The dielectric constant is a measure of a material's ability to reduce the electric field within it, essentially indicating how easily the material can be polarized by an electric field. The estimated value of ε = 2 is quite low, implying that the binding site does not easily polarize in response to an electric field. Materials with a low dielectric constant absorb microwaves less efficiently because of their reduced ability to interact with the oscillating electric field of microwaves. This reduced interaction means that they convert microwave energy into heat less effectively. Another explanation could be the duration of interaction with the solvent, which was 9 times lower in microwave extraction than in traditional extraction, so extraction could not be completed completely. The yields of chlorophyll extracted from spinach byproducts via conventional green extraction and supercritical extraction methods were determined in consecutive studies, and the highest yields were 96% and 50% for conventional and supercritical extraction methods, respectively (Derrien et al., 2017 , 2018 ). The chlorophyll contents of green gooseberry plants extracted via conventional and ultrasonic-assisted extraction were analyzed, and the highest extraction yield was obtained via traditional extraction (Hussain et al., 2023 ). One of the studies on chlorophyll extraction from microalgae via traditional, microwave and supercritical fluid extraction methods reported that a relatively high chlorophyll content was obtained via microwave extraction (Georgiopoulou et al., 2023 ). On the other hand, another study compared traditional, microwave and pressurized liquid extraction methods, and the extraction yield and extraction amount were lower for microwave extraction (Gilbert-López et al., 2017 ). The recovery of pigments from a macroalgae, Ulva rigida , by traditional, microwave-assisted and ultrasound-assisted extraction methods was found to be statistically similar in another study (Martins et al., 2021 ). Even though the results seem contradictory, the nature of the samples and solvents, appliance used in the studies and process conditions might have significantly affected the results. 3.2. Electrospinnability and Properties of the Feed Solutions The feed solution properties help explain electrospinnability. The electrical conductivity, surface tension and viscosity of the solutions are presented in Table 3 . The electrical conductivity of the solution should be greater than zero because only conductive polymers have a high repulsion force to overcome the surface tension of a droplet to form a nanofiber (Wang et al. 2013 ).The highest conductivity was measured for the zein solution (0.90±0.07 mS/cm). The addition of chlorophyll to the zein solution decreased the conductivity. Furthermore, there was no significant difference between solutions prepared with different chlorophyll extracts. However, the solutions prepared with Eu (Solutions 6 and 7) were less conductive than the other solutions were ( p < 0.05). The conductivity of both synthetic and natural Eu has been documented to range from 10⁻¹³ to 10⁻⁵ S/cm (Meredith & Sarna, 2006 ; Osak et al., 1989 ) and this variation can be explained by the measurement conditions, especially the humidity in the environment (Jastrzebska et al., 1995 ). The fact that the solution prepared with Eu is less conductive can be explained by the quinones and hydroquinones found in the structure of eumelanins. These reduced and oxidized components give melanins semiconductor properties. Owing to their biocompatible and biodegradable semiconductor properties, the Eu pigment, which is an organic semiconductor, has the potential to be used in the field of organoelectronics (Matos-Peralta et al., 2023 ). The mildly conductive property of Eu might affect the conductivity and cause less conductive last solutions. While zein and zein with chlorophyll extract solutions produced by uniaxial electrospinning were successfully electrospun and resulted in unbeaded nanofiber formations, nanofiber formations were not obtained with solutions containing Eu (Solutions 6 and 7), which presented the lowest conductivity results. The lower electrical conductivity of the solutions might have caused the beaded structure in this study. To achieve nanofiber morphology by electrospinning, the repulsion force should surpass the surface tension. When the surface tension exceeds a certain threshold, droplet formation occurs instead of the desired nanofiber structure (Ki et al., 2005 ). The surface tension of the solutions was measured the same statistically for solution prepared with different chlorophyll extracts and zein ( p < 0.05). Higher surface tension results were measured for solutions with Eu (Solutions 6 and 7). The surface tension of water is greater than the surface tension of dimethyl sulfoxide (DMSO). The surface tension of DMSO at 20°C is 43.5 mN/m, and the surface tension of water is 72.8 mN/m at 20°C (Kalová & Mareš, 2022 ; Zhao et al., 2021 ). In this study, an 80% ethanol solution was used, and its surface tension was measured as 24.8 mN/m at 20°C (Organisation Internationale de Métrologie Légale, 1975 ). According to these measurements, the surface tension of the 80% ethanol solution is lower than that of DMSO, and the higher surface tension of EU1 and EU2 could be explained in this manner. Additionally, the negatively charged eumelanin pigment may have increased the surface tension of the DMSO solution. The second assumption explaining the high surface tension of the Eu/DMSO solution may be that this solution was prepared at a low concentration. On the basis of these results, when we evaluate the eumelanin/DMSO solution, it can be said that eumelanin prepared in a DMSO solution is a disadvantageous product in electrospinning applications. Nanofiber formation was not achieved with Solutions 6 and 7 (Fig. 1 i and j). High surface tension could affect the morphology of the electrospun samples. Viscosity is a crucial parameter in the electrospinning process. Notably, all the solutions displayed Newtonian characteristics, and all the solutions with extracts were found to be significantly the same except for EU1 and EU2 ( p < 0.05). The viscosity of the zein solution was lower than that of the zein and chlorophyll solutions. The interaction between the extracts and zein might cause an increase in viscosity. A previous study reported that there is a direct proportional relationship between the viscosity of a solution and the average diameter of ultrathin fibers (İnanç Horuz & Belibağlı, 2018 ). According to the results of this study, as the viscosity of the feed solution increased, the average diameter decreased. The thinnest nanofibers (557.90 ± 129.41 and 652.80 ± 151.10 nm) were obtained from the solutions with the highest viscosity (0.15 ± 0.01 mPa.s). The lowest viscosity value was obtained for Solution 7 (0.05±0.01 Pa.s). The lower viscosity values were probably due to the lower zein concentrations used in Solutions 6 and 7. An increase in viscosity promotes the formation of smooth, bead-free fibers (Lu et al., 2016 ). The entanglement of molecules and viscosities of EU1 and EU2 were not enough for nanofiber formation. 3.3. Characterization of the electrospun samples 3.3.1. Zeta potential measurements The zeta potentials of the electrospun samples were measured, and the results are shown in Table 3 . All of the electrospun samples had negative zeta potentials except zein. The zeta potential of samples prepared via chlorophyll extracts derived from microwave-assisted extraction (FMWU and WMWU) was greater than that of their counterparts prepared from extracts obtained via traditional extraction methods (FTU and WTU). A similar pattern was also observed with the coaxial electrospinning method. To maintain the suspension in a stable and dispersed state, the zeta potential values should exceed + 25 mV or fall below − 25 mV (Anonymous, 1999 ). According to the results, the electrospun samples produced with fresh and waste spinach extracts by microwave extraction were more stable than the other samples prepared with extracts obtained via traditional extraction. The EU 1 and EU 2 samples were prepared with WMWE, and their zeta potential values were lower than those of WMWU ( p < 0.05). The addition of Eu and a lower amount of zein might lead to a decrease in the zeta potential. After the addition of chlorophyll extracts into the nanofiber system, the zeta potential of the nanofibers transitioned from a positive charge to a negative charge. Considering that the zeta potential is a measurement of the electrical repulsive forces between particles and is an expression of surface charge, reconfiguration of the surface coverage and greater unfolding of anionic groups could explain the measurement results (Agarry, Wang, Cai, Kan, et al., 2022). The change in zeta potential was greater in the microwave samples than in the traditional samples for both electrospinning methods. Therefore, the extraction method has a significant effect on the morphology of the nanofibers. Table 3 Zeta potential, diameter and encapsulation efficiency of the electrospun samples Sample no/code Zeta potential (mV)* Nanofiber diameter (nm) Encapsulation efficiency (%)** DPPH (mg trolox/g dry weight)*** Chlorophyll a Chlorophyll b 1 0.16±0.03 h 905.72 ± 233.30 bc - - - 2 -0.47±0.10 bc 899.18 ± 212.34 bc 79.31±0.15 h 77.99±2.10 D 21.72 ± 0.47 e 3 -0.37±0.11 cd 865.60 ± 191.65 bc 63.43±0.07 e 82.95±0.04 E 16.02 ± 0.44 d 4 -0.68±0.05 a 557.90 ± 129.41 ab 70.75±0.25 f 72.16±0.10 C 21.95 ± 0.22 e 5 -0.68±0.05 a 652.80 ± 151.10 ab 63.50±0.21 e 57.18±0.74 A 21.07 ± 0.29 e 6 -0.52±0.11 b 566.46 ± 207.00 ab 61.37±0.24 d 61.45±0.97 B 25.49 ± 0.53 f 7 -0.35±0.01 de - 57.13±1.27 c 70.48±1.37 C 30.46 ± 0.51 h 8 -0.17±0.01 fg 341.68 ± 106.55 a 53.50±0.77 b 126.63±0.48 H 8.54 ± 0.13 ab 9 -0.13±0.03 g 303.52 ± 86.75 a 61.59±0.19 d 121.38±1.96 G 8.08 ± 0.20 a 10 -0.25±0.03 ef 1061.16 ± 319.85 c 75.47±0.27 g 124.39±4.03 GH 10.81 ± 0.36 c 11 -0.32±0.05 de 309.32 ± 100.37 a 49.53±0.36 a 88.58±2.01 F 9.83 ± 1.17 bc FTE - - - - 37.73±1.39 i WTE - - - - 28.84±0.52 g FMWE - - - - 49.86±2.78 j WMWE - - - - 25.31±0.33 f *Data are displayed as the mean ± standard deviation of three replicates. Different letters indicate statistically significant differences ( p < 0.05). ** Data are displayed as the mean ± standard deviation of three replicates. The lowercase and uppercase letters indicate significant differences in chlorophyll a and b , respectively ( p < 0.05). *** Data are displayed as the mean ± standard deviation of three replicates. Different letters indicate significant differences ( p < 0.05). 3.3.2. SEM The surface morphologies of the samples are shown in Fig. 1 . SEM analysis revealed that all the samples had fibrous structures except EU1 and EU 2 (Fig. 1 i and j). In addition, samples electrospun by coaxial electrospinning presented lower average diameter results but beady fiber formation. While EU 1 represented relatively thin but beady fiber formation, no fiber formation was observed for EU2. The average diameters of zein (k) as a control and samples containing chlorophyll extracts (a, b, c, d and i) electrospun by uniaxial electrospinning were calculated as 905.72 ± 233.30, 899.18 ± 212.34, 865.60 ± 191.65, 557.90 ± 129.41, 652.80 ± 151.10 and 566.46 ± 207 nm, respectively. Nanofibers produced with microwave extracts (FMWE and WMWE) resulted in the formation of thinner fibrous nanofibers (Fig. 1 c and d). Even though these samples had higher viscosity values than the samples prepared with extracts obtained via traditional extraction, FMWE had the highest conductivity and the lowest surface tension values measured for WMWE (Table 3 ). These properties could lead to the formation of thinner nanofibers. Compared with the other samples, the EU 1 and EU 2 samples presented the lowest conductivity and viscosity and the highest surface tension values and were prepared with lower zein concentrations. It is known that insufficient concentrations of zein result in bead formation due to the unavoidable occurrence of Rayleigh instability (Chang et al., 1999 ). In addition, high surface tension could cause bead formation in the EU 1 and EU 2 samples (Fong et al., 1999 ). In coaxial electrospinning, all the samples were in beady formation. Insufficient entanglement of polymers can result in the formation of beads or droplets during the electrospinning process (Kriegel et al., 2009 ). Zein solution was sent to the system separately in coaxial electrospinning and the viscosity of the zein solution without chlorophyll might result in bead and beaded nanofiber formation. FTU, WTU, FMWU and WMWU: Electrospun samples prepared with FTE, WTE, FMWE and WMWE, respectively via uniaxial electrospinning; EU 1&2: Electrospun samples prepared with eumelanin and WMWE via uniaxial electrospinning; FTC, WTC, FMWC and WMWC: Electrospun samples prepared with FTE, WTE, FMWE and WMWE, respectively via coaxial electrospinning. 3.3.3. FTIR measurements The infrared spectra of the chlorophyll extracts and nanofibers are depicted in Fig. 2 . The observed peaks in the chlorophyll extracts are consistent with previous studies (Kang et al., 2019 ; Sengupta et al., 2015 ; Yang et al., 2022 ; Zhang et al., 2019 ). The broad absorption observed at 3305 cm⁻¹ is attributed to the O–H stretching vibration of ethanol. The sharp peak at 2971 cm⁻¹ could be indicative of the C–H stretching vibration of chlorophyll. The peaks at 2878 and 2880 cm⁻¹ are caused by asymmetric and symmetric CH 2 and CH 3 stretching. The pronounced peak at 1640 cm⁻¹ is consistent with C = O bonding, and the peaks observed in the ranges of 1045–1100 cm⁻¹ and 1377 cm⁻¹ could be attributed primarily to the C–C and C–N vibrations of the tetrapyrrole ring structure of chlorophyll. The characteristic zein spectrum has an −OH group at 3340 cm − 1 , aliphatic C−H stretching bands at 2960 and 2868 cm − 1 , an amide peak Ι at ∼1650 cm − 1 , an amide ΙΙ at 1530 and 1515 cm − 1 , an amide ΙΙΙ at 1240 cm − 1 and C−N stretching at 1445 cm − 1 (Bumedi et al., 2023 ; Dehcheshmeh & Fathi, 2019 ; Sessa & Woods, 2011 ). The absorbance bands of zein nanofiber were observed for −OH group at 3284 cm − 1 , carboxylic acids at 2958 and 2873 cm − 1 , amide Ι (C=O stretching) at 1644 cm − 1 , amide ΙΙ (N−H bending) at 1531 and 1519 cm − 1 , amide ΙΙΙ (C−N stretching) at 1241 cm − 1 and C−N stretching at 1446 cm − 1 . The spectrum of the chlorophyll-loaded nanoparticles underwent some changes. Compared with that of the extracts, the spectrum of the chlorophyll-loaded nanoparticles exhibited a downward shift from 3305 to 3285 cm − 1 . Furthermore, compared with the peak intensities of the extracts in the wavenumber range of 2800–3000 cm − 1 , a reduction in the peak intensities was observed for the nanoparticles. The observed decrease in the vibrations of the methylene and methyl groups might be attributed to the interaction between the chlorophyll phytol group, which serves as the core, and the hydrophobic regions of zein, which act as wall (Ledri et al., 2024 ; Zhang et al., 2020 ). The impact of incorporating chlorophyll extracts into zein is most evident particularly in the −OH group (3284 cm − 1 ), the amide I (1644 cm − 1 ) and amide II (1531 and 1519 cm − 1 ) regions. The observed changes in these regions may be attributed to the hydrophobic interaction between chlorophyll and zein (Agarry et al., 2023 ). While EU 1 and EU 2 exhibited similar spectra, alterations were detected in specified regions compared with WMWU. The Eu samples showed downward shift from 3285 to 3282 cm − 1 , 1645 to 1644 cm − 1 , 1520 to 1519 cm − 1 , 1447 to 1446 cm − 1 and 1241 to 1240 cm − 1 . The addition of Eu to the electrospun samples might interact with zein and chlorophyll and cause alterations in the spectra. Moreover, the intensities of these peaks were greater in samples electrospun by uniaxial electrospinning than in those electrospun by coaxial electrospinning. The formation of an interaction between the core and the wall could be inferred by observing changes in the intensity of the bands or the appearance of new bands in the electrospun samples in comparison to the samples of extracts and zein nanofiber. Consequently, successful encapsulation via uniaxial and coaxial electrospinning techniques was confirmed. 3.3.4. DSC measurements The thermal properties of the extracts, zein and nanofibers were examined via DSC, and the results are shown in Fig. 3 . As shown in Fig. 3 a, endothermic peaks were observed for all the chlorophyll extracts. The chlorophyll extracts obtained via traditional extraction (FTE and WTE) presented similar profiles, whereas FTE had endothermic peaks at 130.25 and 134.8 °C, and peaks at 138.49 and 143.33 °C were observed in the WTE sample. According to several studies (Agarry, Wang, Cai, Kan, et al., 2022; Kang et al., 2019 ; Ledri et al., 2024 ), these peaks could be the melting points of chlorophylls, which are known to melt at 115–133 °C. The samples extracted via microwave extraction (FMWE and WMWE) presented peaks at 101.53–107.5 °C, which can be related to the loss of bound water. Similar results were reported in a previous study (Agarry, Wang, Cai, Kan, et al., 2022). Similar peaks at 122.86-136.22 °C and 128.83-142.47 °C, as observed in the FTE and WTE, could be related to chlorophyll. As reported in previous studies, carotenoids melt within the range of 175–200 °C (Sy et al., 2012 ), and since no peak within this range was observed in any of the extracts, it can be inferred that a selective extraction process for chlorophyll was applied during both traditional and microwave-assisted extraction procedures. Zein showed an endothermic peak at 144.18 °C, and after electrospinning, the peak shifted to 158.39 °C in the zein nanofiber (Fig. 3 b). The highest degradation temperatures were detected for EU 1 (170.62 °C) and EU 2 (169.93 °C). While peaks were observed between 159.82 and 162.37 °C in the electrospun samples obtained via uniaxial electrospinning, the degradation temperatures were measured between 149.58 and 157.83 °C for the samples obtained via coaxial electrospinning (Fig. 3 b). Nanostructures containing chlorophyll extracts did not show any additional peak; therefore, it can be stated that chlorophyll is compatible with zein. Comparable results have also been reported in a previous study (Agarry, Wang, Cai, Kan, et al., 2022). The DSC assay results demonstrated that chlorophyll was successfully encapsulated with zein by electrospinning. The chlorophyll extracts encapsulated with zein presented higher degradation temperatures than did the chlorophyll extracts and E141 (ii), indicating the thermal protection provided by the electrospinning method. In addition, because the temperatures of degradation were lower for the coaxial electrospinning samples than for the uniaxial electrospinning samples, it can be claimed that uniaxial electrospinning enables better thermal protection than coaxial electrospinning. This result could be the result of bead formation in the coaxial electrospinning samples. All of our samples electrospun by coaxial electrospinning showed beaded fiber formations (Fig. 1 e,f,g and h). In a previous study the presence of beads within the fiber mats significantly compromised their structural integrity and resulted in increased fragility. Consequently, this weak lamination caused the beaded fibers to be more prone to breakage. This increased susceptibility to fracture may have also decreased the thermal stability of the samples. Additionally, based on the results, the incorporation of Eu into the system improved the thermal stability of chlorophyll. 3.4. Encapsulation Efficiency of Electrospun Samples The encapsulation efficiencies of the electrospun samples produced via uniaxial electrospinning ranged from 57.13 to 82.95%. In uniaxial electrospinning, wall and core materials are mixed and delivered through the same tip into the electrical field. Therefore, the encapsulation efficiency can be affected by the interaction between the wall and core materials. The hydrophobic interactions between the nonpolar regions of zein and the hydrophobic regions of chlorophyll molecules could impact the stability of the chlorophyll molecules within the nanofibers. According to previous studies, increasing the number of hydrophobic regions and increasing the number of nonpolar interactions and polar bonds of the carbonyl group of the chlorophyll molecule improve the encapsulation efficiency (Agarry et al., 2023 ; Zhang et al., 2020 ). In this study, the highest encapsulation efficiency of chlorophyll a was determined for FTU and the lowest efficiency was measured for EU 2. The lowest values were determined for electrospun samples prepared with waste extracts (EU 2, EU1, WMWU and FMWU). It can be deduced from the data that nanofibers prepared from fresh spinach extracts result in higher encapsulation efficiencies for chlorophyll a . The encapsulation efficiency of chlorophyll b did not follow the same pattern as that of chlorophyll a . While one of the lowest efficiencies was for the WTU for chlorophyll a , the highest efficiency of chlorophyll b was achieved for the WTU. Additionally, higher efficiencies were calculated for the WTU and FMWU samples for chlorophyll b than for chlorophyll a . In a previous study of the microencapsulation of chlorophyll, the encapsulation efficiency of chlorophyll b was also greater than that of chlorophyll a (Dewi et al., 2022 ). Importantly, the extraction technique significantly influences the encapsulation efficiency. FTU and WTU had higher encapsulation efficiencies than FMWU and WMWU for both chlorophyll a and b . Extraction by microwave energy could influence the chemical structure of chlorophyll molecules and the ability to interact between wall material and chlorophyll, resulting in lower encapsulation efficiencies in uniaxial electrospinning. The encapsulation efficiency of the nanofibers prepared via coaxial electrospinning ranged from 49.53-126.64%. The encapsulation efficiency of chlorophyll a was lower than that of uniaxially aligned nanofibers, except for the FMWC sample. Additionally, the efficiencies of chlorophyll b in addition to WMWC were calculated to be higher than 100%, unusually. In coaxial electrospinning, the core and wall materials are fed into the system separately and spun simultaneously. The lower chlorophyll a efficiency and exceptional encapsulation efficiency of chlorophyll b could be related to the conversion of chlorophyll a to chlorophyll b because of the high voltage applied directly to the chlorophyll extracts during processing. 3.5. Antioxidant activity The antioxidant activities of the extracts and electrospun samples were determined via DPPH analysis via a spectrophotometer, and the results are presented in Table 3 . The highest antioxidant activity (49.86±2.78 mg Trolox/g DW) was observed in FMWE for all the samples. Although the highest total chlorophyll content was measured for FTE (19.38 mg/g DW), the chlorophyll b content of FMWE (18.5 mg/g DW) was greater than that of FTE. A relatively high chlorophyll b content may cause greater inhibition of free radicals than chlorophyll a . Hsu et al. also reported that chlorophyll b had slightly greater scavenging activity than chlorophyll b . Furthermore, microwave extraction could improve the antioxidant activity of extracts. Microwave energy initiates cellular disruption within plant materials, facilitating the penetration of solvent into the solid matrix, thereby promoting the dissolution and subsequent release of compounds into the surrounding solvent (Upadhyay et al., 2012 ). When the DSC results were considered, the peaks observed in FMWE and WMWE were also absent in the FTE and WTE samples (Fig. 3 ). Therefore, the extraction method has a substantial effect on the antioxidant activity. The first and second highest DPPH values in the electrospun samples were measured for EU 2 (30.46±0.51 mg Trolox/g DW) and EU 1 (25.49±0.53 mg Trolox/g DW), respectively. Considering the higher antioxidant activities obtained from EU 1 and EU 2 than from WMWU and the higher proportion of Eu in EU 2 than in EU 1, it can be inferred that the addition of Eu into the encapsulation media positively affects the antioxidant activity. Previous studies have confirmed that Eu exhibits antioxidant activity (Bayram et al., 2020 ; Cecchi et al., 2020 ; Song et al., 2023 ). When comparing electrospinning techniques in terms of antioxidant activity, nanofibers fabricated via uniaxial electrospinning showed greater antioxidant activity than those produced by coaxial electrospinning. The core shell structure formed during coaxial electrospinning might hinder the chemical reaction between chlorophyll and the DPPH reagent and lead to decreased antioxidant activity. Furthermore, hydrophobic interactions between zein and chlorophyll molecules in uniaxial electrospinning may enhance the antioxidant activity of nanofibers. 3.6. Light stability The stability of the extracts and nanofibers against light was evaluated, and the retention of chlorophyll after 10 days of exposure to sunlight at room temperature is shown in Fig. 4 . The chlorophyll a content of the extracts decreased sharply on the first day. On day 4, the chlorophyll level decreased to very low levels and no chlorophyll was detected in the extracts thereafter. The retention of chlorophyll a content of nanofibers was ranged from 4–61%. The highest chlorophyll a contents were detected in the microwave samples prepared via uniaxial electrospinning at the end of the test. Only a negligible amount of chlorophyll a was detected in the nanofibers produced by coaxial electrospinning on day 10. Chlorophyll b , like chlorophyll a , could not be detected in the extracts after day 4. Similarly, no chlorophyll b remained in the nanofibers produced by uniaxial electrospinning on day 7. Chlorophyll b was still detected on even day 10 for the nanofibers produced by coaxial electrospinning, although their retention percentages were quite low (18–22%) compared with that of chlorophyll a . In a previous study, better stability against light was expected from Eu-added electrospun samples, because Eu was proposed to possess photoprotective properties (Bayram et al., 2020 ). However, in this study, the addition of Eu did not effectively protect chlorophyll from light. This result could be attributed to the low concentration of the Eu pigment in the solution. According to the results, uniaxial electrospinning improved the stability of chlorophyll a and provided better protection from degradation under light. 3.7. The addition of encapsulated chlorophylls to yogurt The stability of color during storage was evaluated by measuring the L*, a*, and b* values of yogurt samples prepared with extracts, electrospun samples and E141 (ii) the results of which are represented in Fig. 5 . The changes in color of the samples during storage are shown in Fig. 6 . Compared with the control yogurt, yogurt supplemented with chlorophyll extracts, electrospun samples and E141 (ii) presented reduced lightness (L*). The L* values of the chlorophyll extracts incorporated into yogurt (FTE, WTE, FMWE and WMWE) and the yogurt sample prepared with E141 (ii) increased over the storage period (Fig. 5 ). A similar decrease was observed in previous studies (Chen et al., 2012 ; Pires et al., 2018 ; Wijesekara et al., 2022 ), and it has been associated with possible degradation or oxidation of color pigments (Wijesekara et al., 2022 ). In contrast, the L* values of the electrospun samples (FTU, WTU, FMWU, WMWU, FTC, WTC, FMWC and WMWC) increased during storage. As the L* index is strongly influenced by the water content in yogurt, the addition of zein might result in free water on the yogurt surface, resulting in a decrease in the L* value (García-Pérez et al., 2005 ; Jrad et al., 2019 ). The a* values of samples changed significantly (p < 0.5). The -a* value can be used as an indicator of the greenness of a product (R. Wang et al., 2013 ). The -a* values of the samples tended to decrease across all samples at different rates during storage. Major decreases were observed in the extracts (FTE, WTE, FMWE and WMWE) and the changes ranged from ≈40–46% at the end of storage. There was no statistically significant difference between samples with different extracts (p < 0.5). The bright green color faded by the end of the storage period (Fig. 6 ). The reduction in yogurts with electrospun samples ranged from ≈17 to ≈24%. Significant differences were detected in the changes in the -a* values between yogurt samples with extracts and those with electrospun fibers (p < 0.5). Compared with their counterparts prepared via coaxial electrospinning (FTC, WTC, FMWC and WMWC), electrospun samples obtained by uniaxial electrospinning (FTU, WTU, FMWU and WMWU) presented fewer changes in a* values; however, the differences were not significant (p < 0.5). High stability and decreased susceptibility to fading or degradation of synthetic colorants were also observed in this study, and the sample prepared with E141 (ii) exhibited the smallest decrease (≈3%). E141(ii) is produced by adding copper to the product obtained from the saponification of a solvent extract derived from strains of edible plant materials. The incorporation of copper into the chlorophyllin structure stabilizes the vibrant green color, enabling various manufacturing processes and any color alteration during extended storage time (Roca & Pérez-Gálvez, 2024 ). The b* values did not change considerably during the 7 days of storage. Table 4 shows that the lowest total color changes (△E*) were observed in yogurt and E141(ii). The △E* and -a* results revealed similar patterns in electropsun samples. Compared with their counterparts prepared via coaxial electrospinning, samples prepared by uniaxial electrospinning presented smaller color change (p < 0.5). Additionally, the greatest changes were observed for the spinach extracts. These results indicate that better green color retention in yogurt can be achieved by the addition of nanofibers. Chlorophyll is a pH-sensitive compound that is unstable at acidic pH values (3.5-5) (Rodriguez-Amaya, 2016 ). Yogurt is an acidic food (pH 4.0-4.4) (De Souza Oliveira et al., 2011 ); therefore, the chlorophyll in the extracts might degrade when yogurt is added directly into yogurt. When the chlorophyll extracts were encapsulated with zein, the stability of the chlorophyll improved. A previous study on different carrier agents for chlorophyll encapsulation reported that zein-chlorophyll nanoparticles presented significantly greater retention than whey protein isolate and casein against low pH and are recommended for use in acidic foods such as yogurt and pickles (Agarry, Wang, Cai, Wu, et al., 2022 ). Table 4 The total color difference (△E*) of yogurt samples Sample no/code ΔE* 2 2.71±0.35 b 3 2.85±0.07 bc 4 2.49±0.74 b 5 3.46±0.20 c 8 2.60±0.24 b 9 4.16±0.96 d 10 3.98±0.97 d 11 5.10±0.90 d FTE 8.86±0.12 e WTE 9.85±0.11 f FMWE 10.26±0.18 f WMWE 9.60±0.24 f E141 1.24±0.24 a Yogurt 1.04±0.36 a Data are displayed as the mean ± standard deviation of three replicates. Different letters indicate statistically significant differences ( p < 0.05). 4. CONCLUSION Chlorophyll extraction from fresh and waste spinach was performed via traditional and microwave-assisted extraction and comparative analyses were discussed. While higher chlorophyll b contents were obtained through microwave-assisted extraction, traditional extraction resulted in higher chlorophyll a contents. Microwave-assisted extraction can be a promising method for chlorophyll extraction because of its rapidity, cost-effectiveness, environmental friendliness, elevated thermal stability, and enhanced antioxidant activity. According to the results of this study, spinach waste, despite its relatively low chlorophyll content, could serve as a low-cost raw material source for the production of natural green colorants. Chlorophyll extracts were successfully encapsulated via uniaxial and coaxial electrospinning. The microscopy images of the electrospun samples revealed that smooth and unbeaded nanofibers were obtained from all the extracts via uniaxial electrospinning. The electrospinning of chlorophyll extracts with Eu did not result in nanofiber formation. The low conductivity and viscosity and high surface tension of the solutions with Eu could have contributed to this outcome. The successful encapsulation and improved thermal stability of the chlorophyll extracts via the electrospinning method were confirmed through DSC analysis. The results of the DPPH assay revealed that higher antioxidant activity is achieved by microwave-assisted extraction. Additionally, the use of zein and Eu as core materials with electrospinning provides advantages in terms of antioxidant activity. The degradation of chlorophyll extracts when they were exposed to sunlight was determined and enhanced stability was observed in the electrospun samples. Additionally, chlorophyll extracts and electrospun samples were incorporated into yogurt, and it was concluded that the color stability of pH-sensitive chlorophyll molecules was improved by the electrospinning method. Further investigations are warranted to explore the broader implications and potential applications of these findings across related fields. Declarations The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution Beyza Sukran Isik: Conceptualization, Methodology, Formal Analysis, Investigation, Data Curation, Writing- Original Draft, Visualization, Funding acquisition, Sinan Bayram: Methodology, Investigation, Writing - Original Draft, Writing- Review&Editing, Filiz Altay: Conceptualization, Methodology, Writing-Review&Editing, Supervision, Funding acquisition. Funding This work was supported by the Research Fund of the Istanbul Technical University (Project ID: 42890). Data Availability No datasets were generated or analysed during the current study. References Agarry, I. E., Ding, D., Cai, T., Wu, Z., Huang, P., Kan, J., & Chen, K. (2023). Inulin–whey protein as efficient vehicle carrier system for chlorophyll: Optimization, characterization, and functional food application. Journal of Food Science , 88 (8), 3445–3459. https://doi.org/10.1111/1750-3841.16703 Agarry, I. E., Wang, Z., Cai, T., Kan, J., & Chen, K. (2022). Chlorophyll encapsulation by complex coacervation and vibration nozzle technology: Characterization and stability study. Innovative Food Science & Emerging Technologies , 78 , 103017. https://doi.org/10.1016/J.IFSET.2022.103017 Agarry, I. E., Wang, Z., Cai, T., Wu, Z., Kan, J., & Chen, K. (2022). Utilization of different carrier agents for chlorophyll encapsulation: Characterization and kinetic stability study. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5188303","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":372118054,"identity":"3f5d2db8-a801-4e9d-85be-a5cf8c01fe7a","order_by":0,"name":"Beyza Sukran Isik","email":"","orcid":"","institution":"Istanbul Technical University","correspondingAuthor":false,"prefix":"","firstName":"Beyza","middleName":"Sukran","lastName":"Isik","suffix":""},{"id":372118057,"identity":"da4eec17-2ff7-471d-a373-fc1b07414a9f","order_by":1,"name":"Sinan Bayram","email":"","orcid":"","institution":"Bayburt University","correspondingAuthor":false,"prefix":"","firstName":"Sinan","middleName":"","lastName":"Bayram","suffix":""},{"id":372118059,"identity":"70950539-eea0-4d92-aee0-e798909b59e8","order_by":2,"name":"Filiz Altay","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYBACPgY2MC0HodiI0MIGVWXMxgbVwkOslsQG4rWwt6VJ3ag5nN4n32PA8KHsMIO99AECWniOHZPOOXY4t42Nx4BxxrnDDDx8CQS0SKS3Sec2QLQw87YBtRByGUxLOhtIy1/itKQdA2lJAGthJEoLz7Fk65xj6YZtbGkFB3vOpfPwnCGghZ+9zfB2To21vHzz4Y0PfpRZy7H3ENCCAg4wEBEto2AUjIJRMAqIAAA4tzL3pItCPgAAAABJRU5ErkJggg==","orcid":"","institution":"Istanbul Technical University","correspondingAuthor":true,"prefix":"","firstName":"Filiz","middleName":"","lastName":"Altay","suffix":""}],"badges":[],"createdAt":"2024-10-01 15:23:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5188303/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5188303/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71353927,"identity":"80ec9a5c-7819-4766-afcd-34bd6918bdbc","added_by":"auto","created_at":"2024-12-13 15:07:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":498083,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of electrospun samples. FTU (a), WTU (b), FMWU (c), WMWU (d), FTC (e), WTC (f), FMWC (g), WMWC (h), EU1 (i), EU2 (j) and zein (k) samples at 10000x magnification.\u003c/p\u003e\n\u003cp\u003eFTU, WTU, FMWU and WMWU: Electrospun samples prepared with FTE, WTE, FMWE and WMWE, respectively via uniaxial electrospinning; EU 1\u0026amp;2: Electrospun samples prepared with eumelanin and WMWE via uniaxial electrospinning; FTC, WTC, FMWC and WMWC: Electrospun samples prepared with FTE, WTE, FMWE and WMWE, respectively via coaxial electrospinning.\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-5188303/v1/ca31758ec263a9fadd45290e.png"},{"id":71353923,"identity":"715496a0-edc0-4f3c-be05-0e4e689e7d75","added_by":"auto","created_at":"2024-12-13 15:07:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":112393,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of the chlorophyll extracts and nanofibers.\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-5188303/v1/860d4469a12b64c2df7b3b00.png"},{"id":71355071,"identity":"187d5b95-278d-4144-ac21-aed826041c54","added_by":"auto","created_at":"2024-12-13 15:15:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32507,"visible":true,"origin":"","legend":"\u003cp\u003eDSC profiles of extracts (a), zein (b) and nanofibers (b).\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-5188303/v1/6a6cc225a7f9744f9d4fcaac.png"},{"id":71353924,"identity":"70372973-209b-48e1-98d2-460092c694e5","added_by":"auto","created_at":"2024-12-13 15:07:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":89243,"visible":true,"origin":"","legend":"\u003cp\u003eChlorophyll retention after 10 days of exposure to sunlight.\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-5188303/v1/6722972a52a7d725a3859b5e.png"},{"id":71355073,"identity":"40a6d295-0152-4089-8da9-c91774f03a3c","added_by":"auto","created_at":"2024-12-13 15:15:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":63096,"visible":true,"origin":"","legend":"\u003cp\u003eL*, a*, b* values of the samples\u003c/p\u003e","description":"","filename":"Picture5.png","url":"https://assets-eu.researchsquare.com/files/rs-5188303/v1/749552fd8b9b02e818487e16.png"},{"id":71353928,"identity":"d255fcc5-d3b7-4335-ac83-121ff0ec7d88","added_by":"auto","created_at":"2024-12-13 15:07:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":427193,"visible":true,"origin":"","legend":"\u003cp\u003eYogurt samples with extracts, electrospun samples and E141 (ii).\u003c/p\u003e","description":"","filename":"Picture6.png","url":"https://assets-eu.researchsquare.com/files/rs-5188303/v1/55aa5657adbeb8ec599b168a.png"},{"id":71355931,"identity":"acf04d89-02d4-4388-87b2-69797cf42a93","added_by":"auto","created_at":"2024-12-13 15:23:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2658115,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5188303/v1/41d5a594-bd67-4e25-a194-31a1b5111d38.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eElectrospinning Encapsulation of Chlorophylls Microwave-Extracted from Spinach Waste and Utilization as Colouring Agent in Yoghurt\u003c/p\u003e","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eSpinach is a green leafy vegetable and a good source of chlorophyll pigments. While there are more than 100 different chlorophyll structures in nature, chlorophyll \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e are the most abundant in spinach (Murcia et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Scheer, 2006). However, spinach is a highly perishable vegetable, and between 13% and 25% of its waste is produced from damaged leaves and solid residues during the juice production process (Derrien et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Valuable bioactive compounds are still present in wastes, which can make spinach waste a considerable raw material for natural green colorants. The extraction of bioactive compounds from waste via novel methods has become popular especially microwave extraction which is considered a sustainable and green technique, providing an efficient solution for industrial applications (Dai \u0026amp; Mumper, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The application of microwaves results in the rupture of plant cell walls, thereby decreasing the resistance to extraction by enhancing internal diffusion processes (Braga et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChlorophylls are lipid-soluble green pigments present in the plastids of a wide range of plants, algae, and certain bacterial species. There are numerous health advantages of chlorophylls. Chlorophyll has strong antioxidant characteristics and helps prevent cancer, diabetes, cataracts, and heart disease (Murcia et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In recent years, a growing body of scientific literature has demonstrated the significant health benefits associated with chlorophyll consumption, including antimutagenic, and antigenotoxic effects. Moreover, various chlorophyll derivatives have been extensively investigated for their potential in preventing and treating specific conditions, such as cancer, infections, and dermatological disorders (Perez-Galvez et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Owing to their vivid green color, chlorophylls also serve as food colorants and are safely incorporated into various products either in pure form or in complex with copper, such as food, cosmetics and oral hygiene products. However, some countries limit the use of copper complexes of chlorophylls in drugs and foods because of concerns regarding the potential toxicity of copper (Simpson et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). There has been a growing interest in natural food colorants, including chlorophylls. The global market for natural food color was valued at an estimated USD 1.33\u0026nbsp;billion in 2022. Projections indicate that this market will expand at a compound annual growth rate (CAGR) of 8.3% from 2023 to 2030 (Natural Food Color Market Size, Share \u0026amp; Trends Analysis, 2023). However, the instability of natural chlorophyll pigments due to exposure to light, heat, air, and extreme pH conditions limits their use in commercial areas (Hsiao et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This problem can be overcome with encapsulation. There are many studies on the encapsulation of chlorophylls by spray drying (Kang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ledri et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Rajabi et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), freeze drying (Dewi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ledri et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Rajabi et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), fluid bed technology (Yang et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), liposomes (Chu et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), complex coacervation (Agarry, Wang, Cai, Kan, et al., 2022), droplet microfluidics (Hsiao et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and ionic gelation (Tekin \u0026amp; Ersus, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, there is currently no documentation of the encapsulation of natural chlorophyll extracts by electrospinning techniques. Electrospinning is a simple, versatile top-down process that is promising because of its high encapsulation efficiency; improved bioavailability, light, thermal and storage stability; and advanced protection against chemical degradation (Anu Bhushani \u0026amp; Anandharamakrishnan, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Rostamabadi et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, zein was selected as a coating material for electrospinning process. Zein is a type of prolamin storage protein that is typically found within protein bodies located in the endosperm of maize. Zein is recognized as an exceptional material for the electrospinning owing to its biodegradability, flexibility, nontoxicity, and fiber-forming characteristics (Wang et al., \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It was also selected specifically because of its thermal and water stability and high oxygen barrier properties (Deng et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). To improve chlorophyll sensitivity, this study aimed to utilize the melanin pigment in addition to zein. Melanins, heterogeneous polymeric pigments, show high levels of antioxidant activity because of their phenolic and indolic components and contribute to the removal of free radicals through reduction reactions (Altındağ et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). They can absorb low-wavelength, high-energy rays such as gamma rays, X-rays and UV rays and may provide increased photostability to light-sensitive products. In addition, research emphasizes that there is an increase in the thermal stability of polymer products provided by the addition of melanin (Di Mauro et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Shanmuganathan et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, the aim was to extract natural green colorants from spinach waste via different extraction techniques and improve their stability via uniaxial and coaxial electrospinning. The morphology, physicochemistry and stability of the fabricated nanofibers were further investigated, and these materials were subsequently added to yogurt as a coloring agent.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eSpinach was purchased from a local market in Istanbul (Turkiye). Spinach leaves were washed, dried and kept at room temperature. After 4 days, spinach was no longer suitable for consumption and was considered spinach waste.\u003c/p\u003e \u003cp\u003eEthanol (purity of 99.9%) was purchased from Isolab. Liquid chromatography (LC)-grade methanol, acetone and dimethyl sulfoxide (DMSO) were acquired from Merck. Chlorophyll \u003cem\u003ea\u003c/em\u003e, chlorophyll \u003cem\u003eb\u003c/em\u003e and zein were procured from Sigma Aldrich. Alkali salts of copper chlorophyllins (E141 (ii)) were purchased from Alfasol Turkiye.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Methods\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Extraction\u003c/h2\u003e \u003cp\u003eFresh spinach and spinach waste were extracted via two different extraction methods which were traditional and microwave extraction.\u003c/p\u003e \u003cp\u003eFor traditional extraction, chlorophyll was extracted from fresh spinach and spinach waste via a previously described method with some modifications (Zhang et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). One hundred grams of spinach was milled with a grinder for 15 s using deionized water at a ratio of 1:2 (w/v) to remove water-soluble compounds. The resulting precipitate was then extracted with 200 ml of ethanol at 60\u0026deg;C for 30 min in a water bath. The solid and liquid parts were separated. The supernatant was transferred to a beaker, and the precipitate was re-extracted. The extraction mixture was placed in a refrigerator at 4\u0026deg;C overnight and centrifuged at 4000 rpm for 10 min at 4\u0026deg;C to separate the proteins. The supernatant was concentrated with a rotary evaporator at 40\u0026deg;C. Ethanol was added to the obtained solid extract at 0.05 g/mL and the extract was stored at -20\u0026deg;C until further analysis. The chlorophyll extracts extracted from fresh spinach and spinach waste via traditional method were coded as FTE and WTE, respectively.\u003c/p\u003e \u003cp\u003eMicrowave extraction was performed with a microwave oven (Samsung GE83XIAND). Spinach waste and raw spinach were homogenized with a grinder for 15 s with deionized water at a ratio of 1:2 (w/v) to remove water-soluble compounds, and the precipitate was collected as in the traditional method. One hundred grams of spinach was extracted in 1000 ml of ethanol. 100 W was applied for 10 min in the microwave oven. After application, the temperature was measured as 55\u0026deg;C. The solid and liquid parts were separated. The supernatant was incubated at 4\u0026deg;C overnight, centrifuged at 4000 rpm for 10 min at 4\u0026deg;C, and concentrated with a rotary evaporator, similar to the traditional method. Ethanol was added to the obtained solid extract at 0.05 mg/mL, and the mixture was stored at -20 \u003csup\u003eo\u003c/sup\u003eC until further analysis. The chlorophyll extracts extracted from fresh spinach and spinach waste via the microwave method were coded FMWE and WMWE, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Production of Eumelanin (Eu) Pigment\u003c/h2\u003e \u003cp\u003eThe Eu pigment was produced by the \u003cem\u003eStreptomyces parvus\u003c/em\u003e BSB49 strain. The production, polymerization and purification of the Eu pigment were performed according to the procedure outlined by Bayram et al (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3. Uniaxial and coaxial electrospinning encapsulation\u003c/h2\u003e \u003cp\u003eThe electrospinning was conducted with electrospinning equipment (Inovenso NE100, Turkiye) at room temperature. The equipment included a syringe pump (New Era Pump Systems Inc., NE-300, USA), tip of the needle, collector plate and high-voltage power supply (Nanofen, Ankara, Turkiye). The compositions and preparation conditions of the feed solutions are given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Zein was dissolved in 80% ethanol at 400 rpm for 2 h at room temperature. Chlorophyll extracts were mixed with zein solution, and the concentration of zein in the final solution was 30% (w/v) for uniaxial electrospinning. Zein solution was prepared at 30% (w/v) for coaxial electrospinning. The Eu solution was prepared with DMSO at 1 mg/mL. The shell and core materials were mixed before electrospinning via uniaxial electrospinning. The flow rates of the zein solution and chlorophyll extracts are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The nanofibers were fabricated on parchment paper, which was used for covering the collector plate.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComposition of the feed solution, electrospinning conditions and characteristics of the feed solutions\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eSample number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eFeed solutions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" morerows=\"1\" nameend=\"c9\" namest=\"c6\" rowspan=\"2\"\u003e \u003cp\u003eElectrospinning conditions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" morerows=\"1\" nameend=\"c12\" namest=\"c10\" rowspan=\"2\"\u003e \u003cp\u003eFeed solution characteristics*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCore composition\u003c/p\u003e \u003cp\u003e(A)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eShell composition (B)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePolymer solution (w/v)\u003c/p\u003e \u003cp\u003eB1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChlorophyll extract\u003c/p\u003e \u003cp\u003e(v/v)\u003c/p\u003e \u003cp\u003eB2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMixing ratio (v/v)\u003c/p\u003e \u003cp\u003eB1:B2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eElectrospinning type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFlow rate (mL/h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eApplied voltage (kV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDistance to the collector plate (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eElectrical conductivity (mS/cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eSurface tension (mN/m)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eApparent viscosity (mPa.s) at 10 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30% zein (6 g)\u003c/p\u003e \u003cp\u003e80% ethanol (20 mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUniaxial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.90\u0026plusmn;0.07\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e25.37\u0026plusmn;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.12\u0026plusmn;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40% zein (6 g)\u003c/p\u003e \u003cp\u003e80% ethanol (15 mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFTE (5 mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUniaxial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.81\u0026plusmn;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e25.20\u0026plusmn;0.56\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.14\u0026plusmn;0.01\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40% zein (6 g)\u003c/p\u003e \u003cp\u003e80% ethanol (15 mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWTE (5 mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUniaxial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.82\u0026plusmn;0.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e25.45\u0026plusmn;0.53\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.14\u0026plusmn;0.01\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40% zein (6 g)\u003c/p\u003e \u003cp\u003e80% ethanol (15 mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFMWE (5 mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUniaxial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.83\u0026plusmn;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e25.36\u0026plusmn;0.61\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.15\u0026plusmn;0.01\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40% zein (6 g)\u003c/p\u003e \u003cp\u003e80% ethanol (15 mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWMWE (5 mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUniaxial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.79\u0026plusmn;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e24.71\u0026plusmn;0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.15\u0026plusmn;0.01\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20% Zein (6 g)\u003c/p\u003e \u003cp\u003e80% ethanol (30 mL)\u003c/p\u003e \u003cp\u003e+\u003c/p\u003e \u003cp\u003eEu (5 mg)\u003c/p\u003e \u003cp\u003eDMSO (5 mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWMWE (5 mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUniaxial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.73\u0026plusmn;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e29.26\u0026plusmn;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.09\u0026plusmn;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25% zein (6.25 g)\u003c/p\u003e \u003cp\u003e80% ethanol (25 mL)\u003c/p\u003e \u003cp\u003e+\u003c/p\u003e \u003cp\u003eEu (17.5 mg)\u003c/p\u003e \u003cp\u003eDMSO (17.5 mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWMWE (5 mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.5:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUniaxial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.72\u0026plusmn;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e29.13\u0026plusmn;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.05\u0026plusmn;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFTE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30% zein (6 g)\u003c/p\u003e \u003cp\u003e80% ethanol (20 mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCoaxial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA: 0.3\u003c/p\u003e \u003cp\u003eB: 0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWTE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30% zein (6 g)\u003c/p\u003e \u003cp\u003e80% ethanol (20 mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCoaxial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA: 0.3\u003c/p\u003e \u003cp\u003eB: 0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFMWE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30% zein (6 g)\u003c/p\u003e \u003cp\u003e80% ethanol (20 mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCoaxial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA: 0.3\u003c/p\u003e \u003cp\u003eB: 0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWMWE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30% zein (6 g)\u003c/p\u003e \u003cp\u003e80% ethanol (20 mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCoaxial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA: 0.3\u003c/p\u003e \u003cp\u003eB: 0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eEu: Eumelanin, FTE: chlorophyll extracted from fresh spinach via traditional extraction, WTE: chlorophyll extracted from spinach waste via traditional extraction, FMWE: chlorophyll extracted from fresh spinach via microwave extraction, WMWE: chlorophyll extracted from spinach waste via microwave extraction.\u003c/p\u003e \u003cp\u003e*Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of three replicates. The values in each column with different letters indicate statistically significant differences (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4. Determination of Solution Properties\u003c/h2\u003e \u003cp\u003eTo assess the electrospinnability of the feed solutions, electrical conductivity, surface tension and viscosity were measured in triplicate.\u003c/p\u003e \u003cp\u003eThe electrical conductivity of the feed solutions was determined via a conductometer (WTW LF95, Germany) at room temperature in triplicate for each solution.\u003c/p\u003e \u003cp\u003eThe surface tension of the feed solutions was measured by a tensiometer (Dataphysics DCAT 11 E, Germany) at room temperature in triplicate for each solution.\u003c/p\u003e \u003cp\u003eThe viscosity of each solution was measured with a rheometer (Haake Rheostress 1, Germany) via a plate-plate sensor (D\u0026thinsp;=\u0026thinsp;35 mm, gap\u0026thinsp;=\u0026thinsp;1 mm) at room temperature in triplicate for each solution. The shear rate was between 0.1 and 100 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The results were modeled via software (Haake RheoWin3 Data Manager, Germany) according to the power-law equation:\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\tau\\:=K{\\dot{\\gamma\\:}}^{n}\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;1\u003c/p\u003e \u003cp\u003ewhere τ is the shear stress (Pa), K is the consistency index (Pa.s\u003csup\u003en\u003c/sup\u003e), n is the flow behavior index and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\dot{\\gamma\\:}\\)\u003c/span\u003e\u003c/span\u003e is the shear rate (s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The apparent viscosity (η) of the samples was calculated at 100 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e via the following equation:\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\eta\\:=\\text{K}{\\dot{\\gamma\\:}}^{n-1}\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;2\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.2.5. Characterization of nanofibers\u003c/h2\u003e \u003cp\u003eThe zeta potential of each sample was measured in triplicate via a dynamic light scattering instrument (Malvern Zetasizer Nano ZS, Worcestershire, UK) at room temperature. Distilled water was used as a dispersant. The nanofibers were dispersed into 0.2% (w/v) distilled water.\u003c/p\u003e \u003cp\u003eThe morphologies of the electrospun samples were determined by scanning electron microscope (SEM) (Zeiss Evo LS10, Germany) at high vacuum with an accelerating voltage. Electrospun samples measuring 1 cm\u003csup\u003e2\u003c/sup\u003e were coated with Au-Pd via a sputter coater (Quorum, SC7620). Images were taken at certain magnifications. The mean diameter of the fibers was determined via SEM images via ImageJ software with the DiameterJ plugin (National Institutes of Health, USA). The size distribution was evaluated via OriginLab software.\u003c/p\u003e \u003cp\u003eThe infrared spectra of the samples were obtained via an FTIR spectrometer (Jasco-4000, United Kingdom) with an attenuated total reflection (ATR) unit attached. The spectra of each sample were measured with 64 scans and a resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eScans were conducted over a spectral range of 400\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe thermal properties of the samples were assessed via differential scanning calorimetry (DSC) (Q10, New Castle, USA) in triplicate for each sample. The spinach extracts, zein and synthetic colorant (E141 (ii)) (5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 mg) and nanofibers (3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 mg) were weighed in a DSC aluminum pan. An empty DSC-pan was used as an inert reference. The sample and the reference pans were then placed inside the calorimeter, cooled to 20\u0026deg;C at a speed of 5\u0026deg;C\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and heated to 300\u0026deg;C at a heating speed of 10\u0026deg;C\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.3. HPLC analysis\u003c/h2\u003e \u003cp\u003eThe chlorophyll amount was determined via reversed phase high performance liquid chromatography (HPLC). Analysis was performed via an Agilent 1260 Infinity HPLC system (USA) coupled to a DAD detector (Agilent, G131D, USA). The compounds were separated via an ACE 5 C18 column (150 \u0026times; 4.6 mm), and the column temperature was maintained at room temperature. The injection volume was 10 \u0026micro;L, and the samples were injected with an autosampler (Agilent 1329B, USA). An isocratic method was used, and the mobile phase was water:methanol:acetone (4:36:60, v/v/v). The flow rate was 0.6 ml/min. The eluted peaks were detected at 661 nm (Li et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Chlorophyll standards and extracts were diluted with methanol/dichloromethane (65/35; v/v) (Gleize et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The electrospun samples were first dissolved in methanol/dichloromethane (65/35; v/v), and then the zein was removed with water and acetone before HPLC analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Antioxidant activity\u003c/h2\u003e \u003cp\u003eThe antioxidant activity of the extracts and nanofibers was determined via 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity (DPPH) analysis (Braca et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). A total of 25 mg of nanofibers was dissolved in methanol. 100 \u0026micro;L extracts or nanofiber solutions were mixed with 3 mL of a 0.004% methanol solution of DPPH. After 30 min in the dark, the absorbance was read at 517 nm against methanol (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.96).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Light stability of chlorophylls\u003c/h2\u003e \u003cp\u003eThe extracts and nanofibers were exposed to direct sunlight to determine the stability of the chlorophyll. First, 0.1 g of nanofibers was dissolved in 2 mL of ethanol (80%, v/v). Samples were collected at days 0, 1, 4, 7 and 10. The chlorophyll content was determined according to the aforementioned method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.6. The addition of chlorophylls to yogurt\u003c/h2\u003e \u003cp\u003eThe chlorophyll-loaded electrospun samples (0.1 g) were mixed with yogurt. 5 ml of chlorophyll extracts and synthetic colorant (E141 (ii)) were added into yogurt and homogeneous end products were obtained. A colorimeter (Konica Minolta Chromameter CR-400, Japan) was used to determine the CIE L*, a*, b* values of the samples, where L* is the lightness of color (100\u0026thinsp;=\u0026thinsp;white, 0\u0026thinsp;=\u0026thinsp;black), a* value (+\u0026thinsp;a* = red, -a* = green), and b* value (+\u0026thinsp;b* = yellow, -b\u0026frasl;*= blue). The measurements were conducted at days 0, 1, 3, 5 and 7. The total color difference (△E*) was determined via Eq.\u0026nbsp;3, as outlined in a previous study (Mercali et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The formula is as follows:\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\varDelta\\:{E}^{*}\\:=\\:\\sqrt{{\\left(\\varDelta\\:{a}^{*}\\right)}^{2}\\:+\\:{\\left(\\varDelta\\:{b}^{*}\\right)}^{2}\\:+{\\left(\\varDelta\\:{L}^{*}\\right)}^{2}}\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;3\u003c/p\u003e \u003cp\u003ewhere △E* represents the total color difference, △a* represents the difference between the a* values of the control and the sample, △b* represents the difference in b* values between the control and the sample, and △L* represents the difference in L* values between the control and sample. The synthetic colorant (E141 (ii)) was selected as a control sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical analysis\u003c/h2\u003e \u003cp\u003eIBM SPSS Statistics 28 (Chicago, IL, USA) software was used for the statistical analysis. The data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3). One-way analysis of variance (ANOVA) was conducted via Duncan\u0026rsquo;s multiple range test. Differences between means were considered significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Effect of the Extraction Method on the Chlorophyll Content\u003c/h2\u003e \u003cp\u003eThe chlorophyll \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e contents of fresh and waste spinach extracted via traditional and microwave extraction are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. According to the results, fresh samples contained higher chlorophyll \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e contents than waste samples. While the highest chlorophyll \u003cem\u003ea\u003c/em\u003e content (13.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 mg/g dry weight (DW)) was measured for fresh spinach extracted via the traditional extraction method (FTE), fresh spinach extracted via microwave extraction (FMWE) presented the highest chlorophyll \u003cem\u003eb\u003c/em\u003e content (6.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 mg/g DW).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChlorophyll content of the extracts\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChlorophyll \u003cem\u003ea\u003c/em\u003e (mg/g DW)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChlorophyll \u003cem\u003eb\u003c/em\u003e (mg/g DW)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFTE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWTE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFMWE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWMWE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eFTE: Fresh spinach extracted via traditional extraction, WTE: Spinach waste extracted via traditional extraction, FMWE: Fresh spinach extracted via microwave extraction, WMWE: Spinach waste extracted via microwave extraction\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe data are displayed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations of three replicates. The values in each column with different letters are statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe chlorophyll content of fresh spinach reported in the literature significantly varies. While one of the studies reported chlorophyll \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e contents in spinach at 37.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4 and 13.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 mg/100 g, respectively (Dermesonluoglu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), another study reported that 100 g of spinach contained 103.37\u0026thinsp;\u0026plusmn;\u0026thinsp;3.94 mg of chlorophyll \u003cem\u003ea\u003c/em\u003e and 32.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76 mg of chlorophyll \u003cem\u003eb\u003c/em\u003e (Lee et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Another study revealed 71 different spinach genotypes and reported that the chlorophyll \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e contents ranged from 475.76 to 1196.45 and from 200.91 to 522.60 nmol/g FW, respectively, in fall-grown spinach. The chlorophyll content of spinach was reported to range between 13.61 and 15.82 mg/g for chlorophyll \u003cem\u003ea\u003c/em\u003e and between 9.16 and 10.18 mg/g for chlorophyll \u003cem\u003eb\u003c/em\u003e in a recent study (Nipa et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In spring-grown spinach, the chlorophyll \u003cem\u003ea\u003c/em\u003e content varies from 646.66 to 1400.00 nmol/g, and the chlorophyll \u003cem\u003eb\u003c/em\u003e content fluctuates from 219.26 to 474.46 nmol/g (Hayes et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The phytochemical composition of spinach is affected by climatic conditions during the growing season (de Azevedo-Meleiro \u0026amp; Rodriguez-Amaya, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), nitrogen status (Rorie et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), leaf maturity (Farnham et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), cultivation methods (Koh et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), postharvest processing conditions (Leong \u0026amp; Oey, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Mart\u0026iacute;nez et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Mendelov\u0026aacute; et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), and plant genotypes (Hayes et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kidmose et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). In addition, different extraction conditions and determination methods can lead to different results. Therefore, the chlorophyll content of the spinach extracts determined in this study may differ from the results reported in the literature.\u003c/p\u003e \u003cp\u003eThe chlorophyll contents of the waste samples were lower than those of the fresh samples. Chlorophyll degrades when exposed to light, air and heat, resulting in the loss of phytol side chain or the removal of central Mg\u003csup\u003e2+\u003c/sup\u003e (Simpson et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The lowest chlorophyll \u003cem\u003ea\u003c/em\u003e content (5.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mg/g DW) was detected in spinach waste extracted via microwave extraction (WMWE) and spinach waste extracted via traditional extraction (WTE) presented the lowest chlorophyll \u003cem\u003eb\u003c/em\u003e content (3.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mg/g DW). In this study, spinach was kept at room temperature for 4 days, after which it was considered spinach waste. There are several studies concerning the chlorophyll content of spinach during storage. One of the studies measured the chlorophyll \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e contents of spinach stored at 5\u0026deg;C and 20\u0026deg;C. The chlorophyll \u003cem\u003ea\u003c/em\u003e content decreased from 103. 37\u0026thinsp;\u0026plusmn;\u0026thinsp;3.94 mg/100 g to 24.53\u0026thinsp;\u0026plusmn;\u0026thinsp;4.84 mg/100 g at 20\u0026deg;C. Even though the chlorophyll \u003cem\u003eb\u003c/em\u003e content increased from 32.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76 mg/100 g to 33.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.02 mg/100 g, the content was 42.02\u0026thinsp;\u0026plusmn;\u0026thinsp;2.26 mg/100 g on the first day of storage at 20 \u0026deg;C. The chlorophyll \u003cem\u003eb\u003c/em\u003e content first increased during storage but then started to decrease on day 2 (Lee et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Another study reported that the chlorophyll \u003cem\u003ea\u003c/em\u003e content decreased continually to \u0026asymp;65% of the initial level by day 4 at 25\u0026deg;C. Moreover, the chlorophyll \u003cem\u003eb\u003c/em\u003e content decreased similarly to that of chlorophyll \u003cem\u003ea\u003c/em\u003e but at a lower rate (Yamauchi \u0026amp; Watada, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Similar results were also observed in this study. Although the rates of decrease in chlorophyll \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e changed, a decrease was recorded for both extraction methods.\u003c/p\u003e \u003cp\u003eAccording to the results, the traditional extraction method resulted in a higher chlorophyll \u003cem\u003ea\u003c/em\u003e content than did microwave extraction. These results can be explained in several ways. The most likely explanation could be the conversion of chlorophyll \u003cem\u003ea\u003c/em\u003e to chlorophyll \u003cem\u003eb\u003c/em\u003e because the chlorophyll \u003cem\u003eb\u003c/em\u003e contents of the microwave samples were higher than those of the samples extracted via traditional method. The transformation of chlorophylls \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e, which is also called the chlorophyll cycle, is believed to yield either chlorophyll \u003cem\u003ea\u003c/em\u003e or \u003cem\u003eb\u003c/em\u003e, according to particular physiological needs (R\u0026uuml;diger, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The metabolism of chlorophyll \u003cem\u003eb\u003c/em\u003e plays a crucial role in the mechanisms of light acclimation in plants (Hu et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Chlorophyll \u003cem\u003ea\u003c/em\u003e is crucial for photochemical processes, whereas chlorophyll \u003cem\u003eb\u003c/em\u003e enhances a plant's ability to capture a broader spectrum of light, particularly because it has strong absorption at approximately 450 nm, a wavelength that chlorophyll \u003cem\u003ea\u003c/em\u003e does not absorb efficiently. This characteristic makes chlorophyll \u003cem\u003eb\u003c/em\u003e particularly important for increasing a plant's light-harvesting ability (Larkum et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Furthermore, the biosynthesis and degradation of chlorophyll \u003cem\u003eb\u003c/em\u003e are closely associated with the assembly and disassembly of light-harvesting complexes, which can be regulated according to varying light conditions (Tanaka \u0026amp; Tanaka, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Vegetables continue to undergo biochemical processes due to their modular structure and ability to maintain physiological autonomy once harvested and placed on supermarket shelves. One of the studies investigated whether endogenous rhythms persist in commercial vegetables and, if so, how these cycles might influence their nutritional quality. It was observed that Chl a/b ratios oscillated under both constant light and darkness in packaged rocket leaves, whereas oscillations in total Chl a\u0026thinsp;+\u0026thinsp;b were detected exclusively under darkness conditions (De Larrinaga et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The conversion could also be triggered during microwave extraction.\u003c/p\u003e \u003cp\u003eThe chlorophyll structure consists of four pyrrole groups linked by a central magnesium ion to form a tetrapyrrole (or porphyrin) ring system, with a 20-carbon hydrocarbon side chain known as a phytol group. The central magnesium atom of chlorophyll may be sensitive to microwaves as a result of the ionic conduction mechanism (Yan et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Interactions between the central magnesium atom and microwaves may cause localized heating or changes in the electronic structure around the magnesium atom, potentially affecting the stability of the chlorophyll molecule.\u003c/p\u003e \u003cp\u003eThe chlorophyll binding site is situated in a predominantly hydrophobic environment, with an estimated dielectric constant (ε) of approximately 2, which is representative of the typical optical dielectric constant observed in organic molecules (Krishtalik et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). The dielectric constant is a measure of a material's ability to reduce the electric field within it, essentially indicating how easily the material can be polarized by an electric field. The estimated value of ε\u0026thinsp;=\u0026thinsp;2 is quite low, implying that the binding site does not easily polarize in response to an electric field. Materials with a low dielectric constant absorb microwaves less efficiently because of their reduced ability to interact with the oscillating electric field of microwaves. This reduced interaction means that they convert microwave energy into heat less effectively. Another explanation could be the duration of interaction with the solvent, which was 9 times lower in microwave extraction than in traditional extraction, so extraction could not be completed completely.\u003c/p\u003e \u003cp\u003eThe yields of chlorophyll extracted from spinach byproducts via conventional green extraction and supercritical extraction methods were determined in consecutive studies, and the highest yields were 96% and 50% for conventional and supercritical extraction methods, respectively (Derrien et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The chlorophyll contents of green gooseberry plants extracted via conventional and ultrasonic-assisted extraction were analyzed, and the highest extraction yield was obtained via traditional extraction (Hussain et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). One of the studies on chlorophyll extraction from microalgae via traditional, microwave and supercritical fluid extraction methods reported that a relatively high chlorophyll content was obtained via microwave extraction (Georgiopoulou et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). On the other hand, another study compared traditional, microwave and pressurized liquid extraction methods, and the extraction yield and extraction amount were lower for microwave extraction (Gilbert-L\u0026oacute;pez et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The recovery of pigments from a macroalgae, \u003cem\u003eUlva rigida\u003c/em\u003e, by traditional, microwave-assisted and ultrasound-assisted extraction methods was found to be statistically similar in another study (Martins et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Even though the results seem contradictory, the nature of the samples and solvents, appliance used in the studies and process conditions might have significantly affected the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Electrospinnability and Properties of the Feed Solutions\u003c/h2\u003e \u003cp\u003eThe feed solution properties help explain electrospinnability. The electrical conductivity, surface tension and viscosity of the solutions are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe electrical conductivity of the solution should be greater than zero because only conductive polymers have a high repulsion force to overcome the surface tension of a droplet to form a nanofiber (Wang et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).The highest conductivity was measured for the zein solution (0.90\u0026plusmn;0.07 mS/cm). The addition of chlorophyll to the zein solution decreased the conductivity. Furthermore, there was no significant difference between solutions prepared with different chlorophyll extracts. However, the solutions prepared with Eu (Solutions 6 and 7) were less conductive than the other solutions were (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05). The conductivity of both synthetic and natural Eu has been documented to range from 10⁻\u0026sup1;\u0026sup3; to 10⁻⁵ S/cm (Meredith \u0026amp; Sarna, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Osak et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1989\u003c/span\u003e) and this variation can be explained by the measurement conditions, especially the humidity in the environment (Jastrzebska et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). The fact that the solution prepared with Eu is less conductive can be explained by the quinones and hydroquinones found in the structure of eumelanins. These reduced and oxidized components give melanins semiconductor properties. Owing to their biocompatible and biodegradable semiconductor properties, the Eu pigment, which is an organic semiconductor, has the potential to be used in the field of organoelectronics (Matos-Peralta et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The mildly conductive property of Eu might affect the conductivity and cause less conductive last solutions. While zein and zein with chlorophyll extract solutions produced by uniaxial electrospinning were successfully electrospun and resulted in unbeaded nanofiber formations, nanofiber formations were not obtained with solutions containing Eu (Solutions 6 and 7), which presented the lowest conductivity results. The lower electrical conductivity of the solutions might have caused the beaded structure in this study.\u003c/p\u003e \u003cp\u003eTo achieve nanofiber morphology by electrospinning, the repulsion force should surpass the surface tension. When the surface tension exceeds a certain threshold, droplet formation occurs instead of the desired nanofiber structure (Ki et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The surface tension of the solutions was measured the same statistically for solution prepared with different chlorophyll extracts and zein (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05). Higher surface tension results were measured for solutions with Eu (Solutions 6 and 7). The surface tension of water is greater than the surface tension of dimethyl sulfoxide (DMSO). The surface tension of DMSO at 20\u0026deg;C is 43.5 mN/m, and the surface tension of water is 72.8 mN/m at 20\u0026deg;C (Kalov\u0026aacute; \u0026amp; Mareš, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this study, an 80% ethanol solution was used, and its surface tension was measured as 24.8 mN/m at 20\u0026deg;C (Organisation Internationale de M\u0026eacute;trologie L\u0026eacute;gale, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1975\u003c/span\u003e). According to these measurements, the surface tension of the 80% ethanol solution is lower than that of DMSO, and the higher surface tension of EU1 and EU2 could be explained in this manner. Additionally, the negatively charged eumelanin pigment may have increased the surface tension of the DMSO solution. The second assumption explaining the high surface tension of the Eu/DMSO solution may be that this solution was prepared at a low concentration. On the basis of these results, when we evaluate the eumelanin/DMSO solution, it can be said that eumelanin prepared in a DMSO solution is a disadvantageous product in electrospinning applications. Nanofiber formation was not achieved with Solutions 6 and 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei and j). High surface tension could affect the morphology of the electrospun samples.\u003c/p\u003e \u003cp\u003eViscosity is a crucial parameter in the electrospinning process. Notably, all the solutions displayed Newtonian characteristics, and all the solutions with extracts were found to be significantly the same except for EU1 and EU2 (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05). The viscosity of the zein solution was lower than that of the zein and chlorophyll solutions. The interaction between the extracts and zein might cause an increase in viscosity. A previous study reported that there is a direct proportional relationship between the viscosity of a solution and the average diameter of ultrathin fibers (İnan\u0026ccedil; Horuz \u0026amp; Belibağlı, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). According to the results of this study, as the viscosity of the feed solution increased, the average diameter decreased. The thinnest nanofibers (557.90\u0026thinsp;\u0026plusmn;\u0026thinsp;129.41 and 652.80\u0026thinsp;\u0026plusmn;\u0026thinsp;151.10 nm) were obtained from the solutions with the highest viscosity (0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mPa.s). The lowest viscosity value was obtained for Solution 7 (0.05\u0026plusmn;0.01 Pa.s). The lower viscosity values were probably due to the lower zein concentrations used in Solutions 6 and 7. An increase in viscosity promotes the formation of smooth, bead-free fibers (Lu et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The entanglement of molecules and viscosities of EU1 and EU2 were not enough for nanofiber formation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Characterization of the electrospun samples\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1. Zeta potential measurements\u003c/h2\u003e \u003cp\u003eThe zeta potentials of the electrospun samples were measured, and the results are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. All of the electrospun samples had negative zeta potentials except zein. The zeta potential of samples prepared via chlorophyll extracts derived from microwave-assisted extraction (FMWU and WMWU) was greater than that of their counterparts prepared from extracts obtained via traditional extraction methods (FTU and WTU). A similar pattern was also observed with the coaxial electrospinning method. To maintain the suspension in a stable and dispersed state, the zeta potential values should exceed\u0026thinsp;+\u0026thinsp;25 mV or fall below \u0026minus;\u0026thinsp;25 mV (Anonymous, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). According to the results, the electrospun samples produced with fresh and waste spinach extracts by microwave extraction were more stable than the other samples prepared with extracts obtained via traditional extraction. The EU 1 and EU 2 samples were prepared with WMWE, and their zeta potential values were lower than those of WMWU (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05). The addition of Eu and a lower amount of zein might lead to a decrease in the zeta potential.\u003c/p\u003e \u003cp\u003eAfter the addition of chlorophyll extracts into the nanofiber system, the zeta potential of the nanofibers transitioned from a positive charge to a negative charge. Considering that the zeta potential is a measurement of the electrical repulsive forces between particles and is an expression of surface charge, reconfiguration of the surface coverage and greater unfolding of anionic groups could explain the measurement results (Agarry, Wang, Cai, Kan, et al., 2022). The change in zeta potential was greater in the microwave samples than in the traditional samples for both electrospinning methods. Therefore, the extraction method has a significant effect on the morphology of the nanofibers.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eZeta potential, diameter and encapsulation efficiency of the electrospun samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample no/code\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eZeta potential (mV)*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNanofiber diameter (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eEncapsulation efficiency (%)**\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDPPH (mg trolox/g dry weight)***\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChlorophyll a\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChlorophyll b\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.16\u0026plusmn;0.03\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e905.72\u0026thinsp;\u0026plusmn;\u0026thinsp;233.30\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.47\u0026plusmn;0.10\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e899.18\u0026thinsp;\u0026plusmn;\u0026thinsp;212.34\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e79.31\u0026plusmn;0.15\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77.99\u0026plusmn;2.10\u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.37\u0026plusmn;0.11\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e865.60\u0026thinsp;\u0026plusmn;\u0026thinsp;191.65\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e63.43\u0026plusmn;0.07\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e82.95\u0026plusmn;0.04\u003csup\u003eE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.68\u0026plusmn;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e557.90\u0026thinsp;\u0026plusmn;\u0026thinsp;129.41\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e70.75\u0026plusmn;0.25\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e72.16\u0026plusmn;0.10\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.68\u0026plusmn;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e652.80\u0026thinsp;\u0026plusmn;\u0026thinsp;151.10\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e63.50\u0026plusmn;0.21\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e57.18\u0026plusmn;0.74\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.52\u0026plusmn;0.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e566.46\u0026thinsp;\u0026plusmn;\u0026thinsp;207.00\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61.37\u0026plusmn;0.24\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e61.45\u0026plusmn;0.97\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.35\u0026plusmn;0.01\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e57.13\u0026plusmn;1.27\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e70.48\u0026plusmn;1.37\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.17\u0026plusmn;0.01\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e341.68\u0026thinsp;\u0026plusmn;\u0026thinsp;106.55\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53.50\u0026plusmn;0.77\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e126.63\u0026plusmn;0.48\u003csup\u003eH\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.13\u0026plusmn;0.03\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e303.52\u0026thinsp;\u0026plusmn;\u0026thinsp;86.75\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61.59\u0026plusmn;0.19\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.38\u0026plusmn;1.96\u003csup\u003eG\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.25\u0026plusmn;0.03\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1061.16\u0026thinsp;\u0026plusmn;\u0026thinsp;319.85\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75.47\u0026plusmn;0.27\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e124.39\u0026plusmn;4.03\u003csup\u003eGH\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.32\u0026plusmn;0.05\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e309.32\u0026thinsp;\u0026plusmn;\u0026thinsp;100.37\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49.53\u0026plusmn;0.36\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e88.58\u0026plusmn;2.01\u003csup\u003eF\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFTE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37.73\u0026plusmn;1.39\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWTE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e28.84\u0026plusmn;0.52\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFMWE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e49.86\u0026plusmn;2.78\u003csup\u003ej\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWMWE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25.31\u0026plusmn;0.33\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*Data are displayed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of three replicates. Different letters indicate statistically significant differences (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e** Data are displayed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of three replicates. The lowercase and uppercase letters indicate significant differences in chlorophyll \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e, respectively (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e*** Data are displayed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of three replicates. Different letters indicate significant differences (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2. SEM\u003c/h2\u003e \u003cp\u003eThe surface morphologies of the samples are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. SEM analysis revealed that all the samples had fibrous structures except EU1 and EU 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei and j). In addition, samples electrospun by coaxial electrospinning presented lower average diameter results but beady fiber formation. While EU 1 represented relatively thin but beady fiber formation, no fiber formation was observed for EU2.\u003c/p\u003e \u003cp\u003eThe average diameters of zein (k) as a control and samples containing chlorophyll extracts (a, b, c, d and i) electrospun by uniaxial electrospinning were calculated as 905.72\u0026thinsp;\u0026plusmn;\u0026thinsp;233.30, 899.18\u0026thinsp;\u0026plusmn;\u0026thinsp;212.34, 865.60\u0026thinsp;\u0026plusmn;\u0026thinsp;191.65, 557.90\u0026thinsp;\u0026plusmn;\u0026thinsp;129.41, 652.80\u0026thinsp;\u0026plusmn;\u0026thinsp;151.10 and 566.46\u0026thinsp;\u0026plusmn;\u0026thinsp;207 nm, respectively. Nanofibers produced with microwave extracts (FMWE and WMWE) resulted in the formation of thinner fibrous nanofibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and d). Even though these samples had higher viscosity values than the samples prepared with extracts obtained via traditional extraction, FMWE had the highest conductivity and the lowest surface tension values measured for WMWE (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These properties could lead to the formation of thinner nanofibers.\u003c/p\u003e \u003cp\u003eCompared with the other samples, the EU 1 and EU 2 samples presented the lowest conductivity and viscosity and the highest surface tension values and were prepared with lower zein concentrations. It is known that insufficient concentrations of zein result in bead formation due to the unavoidable occurrence of Rayleigh instability (Chang et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). In addition, high surface tension could cause bead formation in the EU 1 and EU 2 samples (Fong et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn coaxial electrospinning, all the samples were in beady formation. Insufficient entanglement of polymers can result in the formation of beads or droplets during the electrospinning process (Kriegel et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Zein solution was sent to the system separately in coaxial electrospinning and the viscosity of the zein solution without chlorophyll might result in bead and beaded nanofiber formation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFTU, WTU, FMWU and WMWU: Electrospun samples prepared with FTE, WTE, FMWE and WMWE, respectively via uniaxial electrospinning; EU 1\u0026amp;2: Electrospun samples prepared with eumelanin and WMWE via uniaxial electrospinning; FTC, WTC, FMWC and WMWC: Electrospun samples prepared with FTE, WTE, FMWE and WMWE, respectively via coaxial electrospinning.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3. FTIR measurements\u003c/h2\u003e \u003cp\u003eThe infrared spectra of the chlorophyll extracts and nanofibers are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe observed peaks in the chlorophyll extracts are consistent with previous studies (Kang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sengupta et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The broad absorption observed at 3305 cm⁻\u0026sup1; is attributed to the O\u0026ndash;H stretching vibration of ethanol. The sharp peak at 2971 cm⁻\u0026sup1; could be indicative of the C\u0026ndash;H stretching vibration of chlorophyll. The peaks at 2878 and 2880 cm⁻\u0026sup1; are caused by asymmetric and symmetric CH\u003csub\u003e2\u003c/sub\u003e and CH\u003csub\u003e3\u003c/sub\u003e stretching. The pronounced peak at 1640 cm⁻\u0026sup1; is consistent with C\u0026thinsp;=\u0026thinsp;O bonding, and the peaks observed in the ranges of 1045\u0026ndash;1100 cm⁻\u0026sup1; and 1377 cm⁻\u0026sup1; could be attributed primarily to the C\u0026ndash;C and C\u0026ndash;N vibrations of the tetrapyrrole ring structure of chlorophyll.\u003c/p\u003e \u003cp\u003eThe characteristic zein spectrum has an \u0026minus;OH group at 3340 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, aliphatic C\u0026minus;H stretching bands at 2960 and 2868 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, an amide peak Ι at \u0026sim;1650 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, an amide ΙΙ at 1530 and 1515 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, an amide ΙΙΙ at 1240 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and C\u0026minus;N stretching at 1445 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e(Bumedi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Dehcheshmeh \u0026amp; Fathi, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sessa \u0026amp; Woods, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The absorbance bands of zein nanofiber were observed for \u0026minus;OH group at 3284 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, carboxylic acids at 2958 and 2873 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, amide Ι (C=O stretching) at 1644 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, amide ΙΙ (N\u0026minus;H bending) at 1531 and 1519 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, amide ΙΙΙ (C\u0026minus;N stretching) at 1241 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and C\u0026minus;N stretching at 1446 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe spectrum of the chlorophyll-loaded nanoparticles underwent some changes. Compared with that of the extracts, the spectrum of the chlorophyll-loaded nanoparticles exhibited a downward shift from 3305 to 3285 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Furthermore, compared with the peak intensities of the extracts in the wavenumber range of 2800\u0026ndash;3000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, a reduction in the peak intensities was observed for the nanoparticles. The observed decrease in the vibrations of the methylene and methyl groups might be attributed to the interaction between the chlorophyll phytol group, which serves as the core, and the hydrophobic regions of zein, which act as wall (Ledri et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe impact of incorporating chlorophyll extracts into zein is most evident particularly in the \u0026minus;OH group (3284 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), the amide I (1644 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and amide II (1531 and 1519 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) regions. The observed changes in these regions may be attributed to the hydrophobic interaction between chlorophyll and zein (Agarry et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). While EU 1 and EU 2 exhibited similar spectra, alterations were detected in specified regions compared with WMWU. The Eu samples showed downward shift from 3285 to 3282 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1645 to 1644 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1520 to 1519 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1447 to 1446 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1241 to 1240 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The addition of Eu to the electrospun samples might interact with zein and chlorophyll and cause alterations in the spectra. Moreover, the intensities of these peaks were greater in samples electrospun by uniaxial electrospinning than in those electrospun by coaxial electrospinning.\u003c/p\u003e \u003cp\u003eThe formation of an interaction between the core and the wall could be inferred by observing changes in the intensity of the bands or the appearance of new bands in the electrospun samples in comparison to the samples of extracts and zein nanofiber. Consequently, successful encapsulation via uniaxial and coaxial electrospinning techniques was confirmed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.3.4. DSC measurements\u003c/h2\u003e \u003cp\u003eThe thermal properties of the extracts, zein and nanofibers were examined via DSC, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, endothermic peaks were observed for all the chlorophyll extracts. The chlorophyll extracts obtained via traditional extraction (FTE and WTE) presented similar profiles, whereas FTE had endothermic peaks at 130.25 and 134.8 \u0026deg;C, and peaks at 138.49 and 143.33 \u0026deg;C were observed in the WTE sample. According to several studies (Agarry, Wang, Cai, Kan, et al., 2022; Kang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ledri et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), these peaks could be the melting points of chlorophylls, which are known to melt at 115\u0026ndash;133 \u0026deg;C. The samples extracted via microwave extraction (FMWE and WMWE) presented peaks at 101.53\u0026ndash;107.5 \u0026deg;C, which can be related to the loss of bound water. Similar results were reported in a previous study (Agarry, Wang, Cai, Kan, et al., 2022). Similar peaks at 122.86-136.22 \u0026deg;C and 128.83-142.47 \u0026deg;C, as observed in the FTE and WTE, could be related to chlorophyll. As reported in previous studies, carotenoids melt within the range of 175\u0026ndash;200 \u0026deg;C (Sy et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and since no peak within this range was observed in any of the extracts, it can be inferred that a selective extraction process for chlorophyll was applied during both traditional and microwave-assisted extraction procedures.\u003c/p\u003e \u003cp\u003eZein showed an endothermic peak at 144.18 \u0026deg;C, and after electrospinning, the peak shifted to 158.39 \u0026deg;C in the zein nanofiber (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The highest degradation temperatures were detected for EU 1 (170.62 \u0026deg;C) and EU 2 (169.93 \u0026deg;C). While peaks were observed between 159.82 and 162.37 \u0026deg;C in the electrospun samples obtained via uniaxial electrospinning, the degradation temperatures were measured between 149.58 and 157.83 \u0026deg;C for the samples obtained via coaxial electrospinning (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Nanostructures containing chlorophyll extracts did not show any additional peak; therefore, it can be stated that chlorophyll is compatible with zein. Comparable results have also been reported in a previous study (Agarry, Wang, Cai, Kan, et al., 2022).\u003c/p\u003e \u003cp\u003eThe DSC assay results demonstrated that chlorophyll was successfully encapsulated with zein by electrospinning. The chlorophyll extracts encapsulated with zein presented higher degradation temperatures than did the chlorophyll extracts and E141 (ii), indicating the thermal protection provided by the electrospinning method. In addition, because the temperatures of degradation were lower for the coaxial electrospinning samples than for the uniaxial electrospinning samples, it can be claimed that uniaxial electrospinning enables better thermal protection than coaxial electrospinning. This result could be the result of bead formation in the coaxial electrospinning samples. All of our samples electrospun by coaxial electrospinning showed beaded fiber formations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee,f,g and h). In a previous study the presence of beads within the fiber mats significantly compromised their structural integrity and resulted in increased fragility. Consequently, this weak lamination caused the beaded fibers to be more prone to breakage. This increased susceptibility to fracture may have also decreased the thermal stability of the samples. Additionally, based on the results, the incorporation of Eu into the system improved the thermal stability of chlorophyll.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Encapsulation Efficiency of Electrospun Samples\u003c/h2\u003e \u003cp\u003eThe encapsulation efficiencies of the electrospun samples produced via uniaxial electrospinning ranged from 57.13 to 82.95%. In uniaxial electrospinning, wall and core materials are mixed and delivered through the same tip into the electrical field. Therefore, the encapsulation efficiency can be affected by the interaction between the wall and core materials. The hydrophobic interactions between the nonpolar regions of zein and the hydrophobic regions of chlorophyll molecules could impact the stability of the chlorophyll molecules within the nanofibers. According to previous studies, increasing the number of hydrophobic regions and increasing the number of nonpolar interactions and polar bonds of the carbonyl group of the chlorophyll molecule improve the encapsulation efficiency (Agarry et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, the highest encapsulation efficiency of chlorophyll \u003cem\u003ea\u003c/em\u003e was determined for FTU and the lowest efficiency was measured for EU 2. The lowest values were determined for electrospun samples prepared with waste extracts (EU 2, EU1, WMWU and FMWU). It can be deduced from the data that nanofibers prepared from fresh spinach extracts result in higher encapsulation efficiencies for chlorophyll \u003cem\u003ea\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe encapsulation efficiency of chlorophyll \u003cem\u003eb\u003c/em\u003e did not follow the same pattern as that of chlorophyll \u003cem\u003ea\u003c/em\u003e. While one of the lowest efficiencies was for the WTU for chlorophyll \u003cem\u003ea\u003c/em\u003e, the highest efficiency of chlorophyll \u003cem\u003eb\u003c/em\u003e was achieved for the WTU. Additionally, higher efficiencies were calculated for the WTU and FMWU samples for chlorophyll \u003cem\u003eb\u003c/em\u003e than for chlorophyll \u003cem\u003ea\u003c/em\u003e. In a previous study of the microencapsulation of chlorophyll, the encapsulation efficiency of chlorophyll \u003cem\u003eb\u003c/em\u003e was also greater than that of chlorophyll \u003cem\u003ea\u003c/em\u003e (Dewi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eImportantly, the extraction technique significantly influences the encapsulation efficiency. FTU and WTU had higher encapsulation efficiencies than FMWU and WMWU for both chlorophyll \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e. Extraction by microwave energy could influence the chemical structure of chlorophyll molecules and the ability to interact between wall material and chlorophyll, resulting in lower encapsulation efficiencies in uniaxial electrospinning.\u003c/p\u003e \u003cp\u003eThe encapsulation efficiency of the nanofibers prepared via coaxial electrospinning ranged from 49.53-126.64%. The encapsulation efficiency of chlorophyll \u003cem\u003ea\u003c/em\u003e was lower than that of uniaxially aligned nanofibers, except for the FMWC sample. Additionally, the efficiencies of chlorophyll \u003cem\u003eb\u003c/em\u003e in addition to WMWC were calculated to be higher than 100%, unusually. In coaxial electrospinning, the core and wall materials are fed into the system separately and spun simultaneously. The lower chlorophyll \u003cem\u003ea\u003c/em\u003e efficiency and exceptional encapsulation efficiency of chlorophyll \u003cem\u003eb\u003c/em\u003e could be related to the conversion of chlorophyll \u003cem\u003ea\u003c/em\u003e to chlorophyll \u003cem\u003eb\u003c/em\u003e because of the high voltage applied directly to the chlorophyll extracts during processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Antioxidant activity\u003c/h2\u003e \u003cp\u003eThe antioxidant activities of the extracts and electrospun samples were determined via DPPH analysis via a spectrophotometer, and the results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe highest antioxidant activity (49.86\u0026plusmn;2.78 mg Trolox/g DW) was observed in FMWE for all the samples. Although the highest total chlorophyll content was measured for FTE (19.38 mg/g DW), the chlorophyll \u003cem\u003eb\u003c/em\u003e content of FMWE (18.5 mg/g DW) was greater than that of FTE. A relatively high chlorophyll \u003cem\u003eb\u003c/em\u003e content may cause greater inhibition of free radicals than chlorophyll \u003cem\u003ea\u003c/em\u003e. Hsu et al. also reported that chlorophyll \u003cem\u003eb\u003c/em\u003e had slightly greater scavenging activity than chlorophyll \u003cem\u003eb\u003c/em\u003e. Furthermore, microwave extraction could improve the antioxidant activity of extracts. Microwave energy initiates cellular disruption within plant materials, facilitating the penetration of solvent into the solid matrix, thereby promoting the dissolution and subsequent release of compounds into the surrounding solvent (Upadhyay et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). When the DSC results were considered, the peaks observed in FMWE and WMWE were also absent in the FTE and WTE samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Therefore, the extraction method has a substantial effect on the antioxidant activity.\u003c/p\u003e \u003cp\u003eThe first and second highest DPPH values in the electrospun samples were measured for EU 2 (30.46\u0026plusmn;0.51 mg Trolox/g DW) and EU 1 (25.49\u0026plusmn;0.53 mg Trolox/g DW), respectively. Considering the higher antioxidant activities obtained from EU 1 and EU 2 than from WMWU and the higher proportion of Eu in EU 2 than in EU 1, it can be inferred that the addition of Eu into the encapsulation media positively affects the antioxidant activity. Previous studies have confirmed that Eu exhibits antioxidant activity (Bayram et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Cecchi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen comparing electrospinning techniques in terms of antioxidant activity, nanofibers fabricated via uniaxial electrospinning showed greater antioxidant activity than those produced by coaxial electrospinning. The core shell structure formed during coaxial electrospinning might hinder the chemical reaction between chlorophyll and the DPPH reagent and lead to decreased antioxidant activity. Furthermore, hydrophobic interactions between zein and chlorophyll molecules in uniaxial electrospinning may enhance the antioxidant activity of nanofibers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Light stability\u003c/h2\u003e \u003cp\u003eThe stability of the extracts and nanofibers against light was evaluated, and the retention of chlorophyll after 10 days of exposure to sunlight at room temperature is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe chlorophyll \u003cem\u003ea\u003c/em\u003e content of the extracts decreased sharply on the first day. On day 4, the chlorophyll level decreased to very low levels and no chlorophyll was detected in the extracts thereafter. The retention of chlorophyll \u003cem\u003ea\u003c/em\u003e content of nanofibers was ranged from 4\u0026ndash;61%. The highest chlorophyll \u003cem\u003ea\u003c/em\u003e contents were detected in the microwave samples prepared via uniaxial electrospinning at the end of the test. Only a negligible amount of chlorophyll \u003cem\u003ea\u003c/em\u003e was detected in the nanofibers produced by coaxial electrospinning on day 10.\u003c/p\u003e \u003cp\u003eChlorophyll \u003cem\u003eb\u003c/em\u003e, like chlorophyll \u003cem\u003ea\u003c/em\u003e, could not be detected in the extracts after day 4. Similarly, no chlorophyll \u003cem\u003eb\u003c/em\u003e remained in the nanofibers produced by uniaxial electrospinning on day 7. Chlorophyll \u003cem\u003eb\u003c/em\u003e was still detected on even day 10 for the nanofibers produced by coaxial electrospinning, although their retention percentages were quite low (18\u0026ndash;22%) compared with that of chlorophyll \u003cem\u003ea\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn a previous study, better stability against light was expected from Eu-added electrospun samples, because Eu was proposed to possess photoprotective properties (Bayram et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, in this study, the addition of Eu did not effectively protect chlorophyll from light. This result could be attributed to the low concentration of the Eu pigment in the solution. According to the results, uniaxial electrospinning improved the stability of chlorophyll \u003cem\u003ea\u003c/em\u003e and provided better protection from degradation under light.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.7. The addition of encapsulated chlorophylls to yogurt\u003c/h2\u003e \u003cp\u003eThe stability of color during storage was evaluated by measuring the L*, a*, and b* values of yogurt samples prepared with extracts, electrospun samples and E141 (ii) the results of which are represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The changes in color of the samples during storage are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eCompared with the control yogurt, yogurt supplemented with chlorophyll extracts, electrospun samples and E141 (ii) presented reduced lightness (L*). The L* values of the chlorophyll extracts incorporated into yogurt (FTE, WTE, FMWE and WMWE) and the yogurt sample prepared with E141 (ii) increased over the storage period (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). A similar decrease was observed in previous studies (Chen et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Pires et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wijesekara et al., \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and it has been associated with possible degradation or oxidation of color pigments (Wijesekara et al., \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In contrast, the L* values of the electrospun samples (FTU, WTU, FMWU, WMWU, FTC, WTC, FMWC and WMWC) increased during storage. As the L* index is strongly influenced by the water content in yogurt, the addition of zein might result in free water on the yogurt surface, resulting in a decrease in the L* value (Garc\u0026iacute;a-P\u0026eacute;rez et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Jrad et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The a* values of samples changed significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.5). The -a* value can be used as an indicator of the greenness of a product (R. Wang et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The -a* values of the samples tended to decrease across all samples at different rates during storage. Major decreases were observed in the extracts (FTE, WTE, FMWE and WMWE) and the changes ranged from \u0026asymp;40\u0026ndash;46% at the end of storage. There was no statistically significant difference between samples with different extracts (p\u0026thinsp;\u0026lt;\u0026thinsp;0.5). The bright green color faded by the end of the storage period (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The reduction in yogurts with electrospun samples ranged from \u0026asymp;17 to \u0026asymp;24%. Significant differences were detected in the changes in the -a* values between yogurt samples with extracts and those with electrospun fibers (p\u0026thinsp;\u0026lt;\u0026thinsp;0.5). Compared with their counterparts prepared via coaxial electrospinning (FTC, WTC, FMWC and WMWC), electrospun samples obtained by uniaxial electrospinning (FTU, WTU, FMWU and WMWU) presented fewer changes in a* values; however, the differences were not significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.5). High stability and decreased susceptibility to fading or degradation of synthetic colorants were also observed in this study, and the sample prepared with E141 (ii) exhibited the smallest decrease (\u0026asymp;3%). E141(ii) is produced by adding copper to the product obtained from the saponification of a solvent extract derived from strains of edible plant materials. The incorporation of copper into the chlorophyllin structure stabilizes the vibrant green color, enabling various manufacturing processes and any color alteration during extended storage time (Roca \u0026amp; P\u0026eacute;rez-G\u0026aacute;lvez, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The b* values did not change considerably during the 7 days of storage.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows that the lowest total color changes (△E*) were observed in yogurt and E141(ii). The △E* and -a* results revealed similar patterns in electropsun samples. Compared with their counterparts prepared via coaxial electrospinning, samples prepared by uniaxial electrospinning presented smaller color change (p\u0026thinsp;\u0026lt;\u0026thinsp;0.5). Additionally, the greatest changes were observed for the spinach extracts.\u003c/p\u003e \u003cp\u003eThese results indicate that better green color retention in yogurt can be achieved by the addition of nanofibers. Chlorophyll is a pH-sensitive compound that is unstable at acidic pH values (3.5-5) (Rodriguez-Amaya, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Yogurt is an acidic food (pH 4.0-4.4) (De Souza Oliveira et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e); therefore, the chlorophyll in the extracts might degrade when yogurt is added directly into yogurt. When the chlorophyll extracts were encapsulated with zein, the stability of the chlorophyll improved. A previous study on different carrier agents for chlorophyll encapsulation reported that zein-chlorophyll nanoparticles presented significantly greater retention than whey protein isolate and casein against low pH and are recommended for use in acidic foods such as yogurt and pickles (Agarry, Wang, Cai, Wu, et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe total color difference (△E*) of yogurt samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample no/code\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eΔE*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.71\u0026plusmn;0.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.85\u0026plusmn;0.07\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.49\u0026plusmn;0.74\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.46\u0026plusmn;0.20\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.60\u0026plusmn;0.24\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.16\u0026plusmn;0.96\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.98\u0026plusmn;0.97\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.10\u0026plusmn;0.90\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFTE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.86\u0026plusmn;0.12\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWTE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.85\u0026plusmn;0.11\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFMWE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.26\u0026plusmn;0.18\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWMWE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.60\u0026plusmn;0.24\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE141\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.24\u0026plusmn;0.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eYogurt\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.04\u0026plusmn;0.36\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eData are displayed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of three replicates. Different letters indicate statistically significant differences (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. CONCLUSION","content":"\u003cp\u003eChlorophyll extraction from fresh and waste spinach was performed via traditional and microwave-assisted extraction and comparative analyses were discussed. While higher chlorophyll \u003cem\u003eb\u003c/em\u003e contents were obtained through microwave-assisted extraction, traditional extraction resulted in higher chlorophyll \u003cem\u003ea\u003c/em\u003e contents. Microwave-assisted extraction can be a promising method for chlorophyll extraction because of its rapidity, cost-effectiveness, environmental friendliness, elevated thermal stability, and enhanced antioxidant activity. According to the results of this study, spinach waste, despite its relatively low chlorophyll content, could serve as a low-cost raw material source for the production of natural green colorants. Chlorophyll extracts were successfully encapsulated via uniaxial and coaxial electrospinning. The microscopy images of the electrospun samples revealed that smooth and unbeaded nanofibers were obtained from all the extracts via uniaxial electrospinning. The electrospinning of chlorophyll extracts with Eu did not result in nanofiber formation. The low conductivity and viscosity and high surface tension of the solutions with Eu could have contributed to this outcome. The successful encapsulation and improved thermal stability of the chlorophyll extracts via the electrospinning method were confirmed through DSC analysis. The results of the DPPH assay revealed that higher antioxidant activity is achieved by microwave-assisted extraction. Additionally, the use of zein and Eu as core materials with electrospinning provides advantages in terms of antioxidant activity. The degradation of chlorophyll extracts when they were exposed to sunlight was determined and enhanced stability was observed in the electrospun samples. Additionally, chlorophyll extracts and electrospun samples were incorporated into yogurt, and it was concluded that the color stability of pH-sensitive chlorophyll molecules was improved by the electrospinning method. Further investigations are warranted to explore the broader implications and potential applications of these findings across related fields.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBeyza Sukran Isik:\u003c/strong\u003e Conceptualization, Methodology, Formal Analysis, Investigation, Data Curation, Writing- Original Draft, Visualization, Funding acquisition, \u003cstrong\u003eSinan Bayram:\u003c/strong\u003e Methodology, Investigation, Writing - Original Draft, Writing- Review\u0026amp;Editing, \u003cstrong\u003eFiliz Altay:\u003c/strong\u003e Conceptualization, Methodology, Writing-Review\u0026amp;Editing, Supervision, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Research Fund of the Istanbul Technical University (Project ID: 42890).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgarry, I. 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Improved CZTSSe thin-film morphology and device performance by using DMSO/DMF blended solvent. \u003cem\u003eApplied Physics A: Materials Science and Processing\u003c/em\u003e, \u003cem\u003e127\u003c/em\u003e(8), 1\u0026ndash;8. https://doi.org/10.1007/S00339-021-04753-W/METRICS\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"chlorophylls, uniaxial electrospinning, coaxial electrospinning, electrospinning encapsulation, spinach waste, microwave extraction","lastPublishedDoi":"10.21203/rs.3.rs-5188303/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5188303/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNatural colorants have become a developing market because of consumer preferences, and more cost-effective and stable production is necessary. In the present study, a natural green colorant was obtained via microwave-assisted extraction from spinach waste to produce affordable, environmentally-friendly and innovative colorants. The extracts were encapsulated with zein via uniaxial and coaxial electrospinning techniques to overcome the stability issues associated with their natural green color. The encapsulation efficiencies of uniaxially encapsulated chlorophyll extracted from spinach waste by microwave and coaxially encapsulated chlorophyll extracted from spinach waste by microwave were 63.50 and 49.53% for chlorophyll \u003cem\u003ea\u003c/em\u003e and 57.18 and 88.58% for chlorophyll \u003cem\u003eb\u003c/em\u003e, respectively. The microwave-assisted extraction and addition of eumelanin significantly improved the antioxidant activity and thermal stability of chlorophylls. The stability test against light revealed that 61% of chlorophyll \u003cem\u003ea\u003c/em\u003e could be preserved by uniaxial electrospinning. In addition, when chlorophyll-loaded nanofibers were incorporated into yogurt, a more stable green color was achieved during storage.\u003c/p\u003e","manuscriptTitle":"Electrospinning Encapsulation of Chlorophylls Microwave-Extracted from Spinach Waste and Utilization as Colouring Agent in Yoghurt","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-13 15:07:49","doi":"10.21203/rs.3.rs-5188303/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-30T08:22:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-26T16:40:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"280386206283825337718110402600931247451","date":"2024-10-14T11:54:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-07T16:44:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"131884762390130074590026979772777345089","date":"2024-10-03T08:43:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-03T08:39:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-02T08:56:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-01T22:41:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food and Bioprocess Technology","date":"2024-10-01T15:19:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"eadb1074-7c97-44c3-abe1-af752204eafb","owner":[],"postedDate":"December 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-12-26T11:38:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-13 15:07:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5188303","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5188303","identity":"rs-5188303","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}