Onions are rich in bioactive and volatile compounds with antioxidant activity. When concentrated in plant extracts and applied directly into food matrices or packaging, they are sensitive to external factors and may have undesirable sensory attributes, such as odors. Therefore, this study sought to encapsulate red onion extract (ROE) in different concentrations (10, 20, 30, and 40%, v/v) into zein fibers by electrospinning. The major volatile compound in ROE was 3(2H)-furanone, 2-hexyl-5-methyl. Adding ROE into the polymeric solutions increased their electrical conductivity and decreased the apparent viscosity, thus decreasing the average diameter of the fibers. The loading capacity of ROE on fibers ranged from 77.3 to 91.5%. The fibers’ random and continuous morphology showed beads at the highest ROE concentration (40%). Fibers membranes showed lower values for contact angles as ROE was added. The antioxidant activity of the fibers ranged from 32.5 to 57.3%.
Research Article
Zein electrospun fibers with red onion extract (Allium cepa L.): Bioactive compounds, thermal stability, hydrophilicity, and antioxidant activity
https://doi.org/10.21203/rs.3.rs-2961211/v1
This work is licensed under a CC BY 4.0 License
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Nanotechnology
Electrospinning
Vegetable extracts
Volatile compounds
Free radicals
Ultrafine fibers
Onion (Allium cepa L.) is a vegetable grown on a large scale almost worldwide. It has medicinal uses, including anti-inflammatory, antioxidant, and antimicrobial activities (Colina-Coca et al., 2017; Jakaria et al., 2019), action against cardiovascular and carcinogenic diseases (Colina-Coca et al., 2017), and other health benefits and functions (Sami, Elhakem, Alharbi, Benajiba et al., 2021). In addition, it has nutrients such as carbohydrates, proteins, vitamins (vitamin C, B6, and folic acid), sugars (arabinose, fructose, glucose, and galactose), minerals (calcium, iron, and sulfur) (Asanga et al., 2015; Sami, Elhakem, Alharbi, Benajiba, et al., 2021), and flavonoids and phenolic components and their glycosylates (Zhao et al., 2021).
Furthermore, onions are rich in volatile compounds and essential minerals, the variety of which aids in maintaining optimal health (Sami, Elhakem, Alharbi, Almatrafi, et al., 2021). Volatile compounds present distinct biological activities (Cecchi et al., 2020). Among the volatiles, sulfur compounds are responsible for the onion taste and odor after cutting and chewing. In addition to volatile sulfur compounds, other polar compounds such as sapogenins, saponins, and flavonoids have been detected in Allium species (Cecchi et al., 2020; Sami, Elhakem, Alharbi, Benajiba, et al., 2021).
One possibility of bioactive and/or volatile compound concentration is plant extract production, which consists of preparations with or without treatment (e.g., techniques such as maceration, infusion, continuous hot extraction, counter-current extraction, microwave-assisted extraction, ultrasound, and supercritical fluid, among others) and/or temperatures and the use of solvents such as water and ethanol, among others (Cruz et al., 2022). Although these compounds have health benefits, they mainly present undesirable odors when concentrated and applied directly to food matrices or packaging (Cecchi et al., 2020; Sami, Elhakem, Alharbi, Almatrafi, et al., 2021). In addition, bioactive compounds are susceptible to external factors such as light, humidity, pH, oxygen, and high temperatures. Therefore, they must be protected to preserve their functionality and stability (Santos et al., 2022; Fonseca et al., 2021). One possibility to protect these compounds from the external environment, whether they are bioactive and/or volatile, is through their encapsulation.
Encapsulation is the process of packaging compounds (e.g., flavor compounds, pigments, acidulants, nutrients, enzymes, and preservatives) into particles, such as fibers and capsules. One emerging encapsulation method is electrospinning, a low-cost and relatively simple technique with high loading capacity. Most importantly, it uses only ambient temperatures in the process, enabling the encapsulation of thermo-sensitive compounds (Fonseca et al., 2020). Recent studies have elucidated that electrospinning is an excellent encapsulation technique, and using zein (protein from corn) as a wall material is highly efficient and capable of producing nanoscale materials (Antunes et al., 2017; Evangelho et al., 2019; Prietto et al., 2018; Radünz et al., 2020, 2021; Santos et al., 2022).
In a previous study by our research group (Cruz et al., 2022), we used red onion skin to produce an extract rich in phenolic compounds, and flavonoids encapsulated into starch ultrafine fibers. To utilize the left onion for other purposes, we decided to prepare and evaluate the onion extract. We hypothesize that preparing a red onion extract (ROE) provides a significant number of bioactive compounds, such as flavonoids, total phenolics, and volatile compounds. Additionally, that encapsulation preserves these compounds from the external environment. Thus, this study aimed to prepare and encapsulate the ROE into zein fibers by electrospinning for possible applications in active packaging or food matrices.
2.1 Material
Red onions (var. ‘Crioula’) were purchased from local businesses in Pelotas (Rio Grande do Sul State, southern Brazil). The materials and chemical reagents used were ethanol (purity ≥ 99.8%, CAS: 64-17-5, Êxodo Científica, Brazil), zein (purity ≥ 97%, CAS: 9010–66 − 6, Brazil), and salicylic acid (CAS: 69-72-7), which were purchased from Sigma Aldrich, USA. Iron sulfate heptahydrate (CAS: 7782-63-0), sodium nitroprusside (CAS: 13755-38-9), and phosphoric acid (CAS: 7664-38-2) were purchased from Merck, Germany. Gallic acid standard (purity > 98%, CAS: 5995–86 − 8) was purchased from Dinâmica, Brazil. Ultrapure water was obtained using the MecLab MegaPurity® system (Jacareí, Brazil). The other reagents used were of analytical grade.
2.2 Methods
2.2.1 Obtaining the red onion extract
Initially, the onions were selected according to the presence of deterioration or apparent fungi, and then the skin was manually removed. The peeled onions were then washed with water, sanitized in aqueous sodium hypochlorite solution (NaClO, 200 ppm, v/w) for 15 min in the proportion of 1 kg of raw material for 2 L of solution, rinsed with filtered water, and dried at room temperature (24 ± 3°C). Afterward, the onions were freeze-dried (Liophilizer, Liotop K108, Brazil) with the following parameters: condenser temperature of -100°C and pressure below 100 µHg.
The ROE preparation was performed according to Sarvinehbaghi et al. (2021), with modifications. The dried onions were ground (Oster, Model OMDR100, China) for approximately 1 min. Subsequently, 35 g of dried onions were mixed in 250 mL of 75% ethanol (in distilled water, v/v) and heated in a thermostatic water bath (Velp Scientifica, Enzymatic Digester-GDE, Italy) at 60°C for 3 h with stirring at 300 rpm. The extractor solution was centrifuged (Eppendorf Centrifuge 5430R, Germany) at 2236 ×g for 10 min at 4 ºC. Next, the supernatant was inserted into a rotary evaporator (Laborota 4000eco, Heidolph, Germany) coupled to an ultra-thermostatic bath (Q214M2, Quimis, Brazil) at 60°C and 90 rpm of agitation until the complete evaporation of the ethanol. Lastly, the liquid ROE obtained was contained in a hermetically sealed amber bottle and stored at -18 ± 2°C until the analysis was carried out.
2.2.2 ROE total phenolic compounds, flavonoids, and anthocyanin content
The content of total phenolic compounds was determined by the Folin-Ciocalteu colorimetric method, according to Swain and Hillis (Swain & Hillis, 1959), with modifications. Gallic acid solutions (0 to 225 µg/mL) were used to construct the calibration curve (R² = 0.999), and the results were expressed in mg gallic acid equivalent (mgGAE/100g).
The total flavonoid content was determined by the aluminum chloride colorimetric method according to the method of Lin and Tang (Lin & Tang, 2007), with adaptations. The method consisted of 1 mL aliquots of ROE mixed with 3 mL of 95% ethanol and 200 µL of aluminum chloride (CAS 7784-13-6) at a concentration of 2.5%; the solution was stirred for 1 min and then left to rest for 40 min in the dark. The absorbance of the sample was measured at 415 nm in a spectrophotometer (Jenway Model 6705 UV Scan/Visible Spectrophotometers, China). Quercetin solutions at concentrations from 0 to 225 µg/mL were used to construct the calibration curve (R² = 0.997), and the results were expressed in mg quercetin equivalents (mgQE/100g).
The total anthocyanin content was determined according to Lee et al. (Lee et al., 2005) using the single pH method, and the absorbance reading of the sample was measured at 520 nm using a spectrophotometer (Jenway Model 6705 Scanning UV/Visible Spectrophotometers, China). The results were calculated according to Eq. 1 and expressed in g cyanidin-3-glycoside equivalents (mgC3GE/100g).
\(\text{Total anthocyanins=}\left(\frac{\text{Abs.*MW* V* DF}}{\text{ε*m*1}}\right)\text{.100}\) Eq. 1.
Where: “Abs” is the absorbance of the sample, “MW” is the molecular weight of cyanidin-3-glycoside (449.2 g/mol), “V” is the volume used for extraction, “DF” is the dilution factor, ε is the molar absorptivity at a wavelength of 520 nm (26,900), “m” is the mass in g of the sample used, and “1” refers to the optical path traveled (quartz cuvette for spectrophotometer).
2.2.3 Volatile composition of ROE headspace by GC-MS (HS-SPME-GC-MS)
The sample containing volatile organic compounds (VOCs) was prepared using the solid phase microextraction method (HS-SPME) and divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber (Supelco, 50/30 µm × 20 mm) preconditioned following the manufacturer’s protocol. To extract the VOCs, 1 mL of red onion extract was placed in a 20 mL flask, followed by adding 1 g of NaCl and 10 µL of standard benzophenone solution (2 µg), sealing the flask using a lid silicone coated with polytetrafluoroethylene. The sealed flasks containing the extract were submerged in a water bath at 40°C for 15 min, and the fibers were then exposed to headspace for 15 min under constant agitation. The VOCs were quantified and identified using a gas chromatography (GC) system (Shimadzu QP2010 UltraPlus, Shimadzu Japan) equipped with a mass detector mass spectrometer (Shimadzu Corporation, Kyoto, Japan) and Rxi-1MS capillary column (30 m × 0.32 mm × 0.25 µm, Restek). The ramp temperature was held at 40°C for 5 min, increased to 50°C at 3°C/min, to 120°C at 5°C/min, to 175°C at 7°C/min, and finally at 230°C at 10°C/min, then maintained under isothermal conditions for 8 min. Mass spectrometry (MS) was operated in full scan mode (m/z mass range 30 to 450). The GC-MS data were analyzed using LabSolution (GCMS solution Version 4.11 SU2). The VOCs were identified by comparing similarity indices and mass spectrum with the National Institute of Standards and Technology (NIST11) system database and retention index calculated from a homologous series of C8-C40 hydrocarbons according to Van Den Dool and Dec. Kratz (1963). Finally, the quantitative analysis was determined by internal standardization. Data were presented through a heat map representing the dominance of compounds (i.e., VOCs) and were grouped using different color combinations. Each colored cell on the map corresponds to a concentration value. The map was drawn using the GraphPad Prism software (version 9.4.1).
2.2.4 Polymeric solution production
Polymeric solutions were prepared to produce fibers by electrospinning. First, the 30% (w/v) zein-based polymeric solutions were prepared with 85% ethanol (v/v, in ultrapure water) and stirred for 30 min for complete solubilization. Next, the polymeric solutions were added with ROE at concentrations of 10, 20, 30, and 40% (v/v) and shaken again for another 15 min to ensure complete homogenization of the extract in the polymeric solution.
2.2.5 Apparent viscosity and electrical conductivity of polymer solutions
Apparent viscosity was determined using a Brookfield digital viscometer with #18 spindle and fixed speed at 100 rpm (Model DV-II, USA) and expressed in cP. The electrical conductivity of the polymer solutions was evaluated using a conductivity meter (EC150 Conductivity Meter, Simpla, China) and expressed in µS/cm. Both analyses were performed in triplicate and at room temperature (22 ± 2°C).
2.2.6 Electrospinning process
To produce the pure zein fibers and zein fibers added with ROE, a horizontal electrospinning station was used, which comprised a high voltage source (Faíscas, Model FA + 30kV, Brazil), a syringe infusion pump (KD Scientific, Model 100, Holliston, England), and a stainless-steel collector covered with aluminum foil to deposit the material. Each polymer solution was individually placed in a 3-mL plastic syringe coupled with a 0.8 mm diameter stainless steel needle. The following parameters were used in the electrospinning process: pumping flow rate of the polymeric solutions of 0.8 mL/h, applied voltage of 20 kV, and 20 cm between the needle tip and the collector. The relative humidity of the air was controlled with a dehumidifier (Relaxmedic, RM-DA0600A, China) at 40 ± 5% and the temperature at 20 ± 2 ºC using air conditioning (Electrolux, VI 12R, Brazil).
2.2.7 Loading capacity
The loading capacity (LC) was determined by spectrophotometry for the content of phenolic compounds and conducted according to Barros et al. (2022). For this, 5 mg of the fibers and 1.5 mL of 75% ethanol (in distilled water, v/v) were homogenized in a vortex for roughly 1 min. Then, the solutions were centrifuged at 8232 x G for 5 min at 4°C. Finally, an aliquot of the supernatant was individually analyzed in a microplate spectrophotometer (SpectraMAX 190, USA) at 725 nm according to the method of Swain and Hillis (1959) adapted for the microplate reader. The LC was expressed as a percentage and calculated according to Eq. 2.
2.2.8 Morphology and diameter
The electrospun fibers morphology was evaluated by scanning electron microscopy (SEM, Jeol, JSM − 6610LV, USA). With double-sided tape, a small portion of the materials was fixed on stubs and covered with gold using a metallizer (Sputtering, Denton Vacuum Desk V, USA). The images were obtained with an acceleration voltage of 15 kV and magnification of 1000 and 6000 x. The mean diameter was determined from the SEM images, and 60 random measurements of each image were performed using the ImageJ® software (version 2015).
2.2.9 Fourier transform infrared spectroscopy
The ROE, zein, and electrospun fibers were individually characterized in a Fourier transform infrared (FTIR) spectrometer (Shimadzu, IR Affinity-1, Japan). Samples were mixed with KBr and pressed into pellets before analysis. The spectra were obtained in the spectral range from 4000 to 400 cm− 1, with a resolution of 2 cm− 1 and 100 scans. The OriginPro® 8.5 program was used to process the data.
2.2.10 Thermogravimetric analysis
For thermogravimetric analysis, ROE, zein, and electrospun fibers were individually packed in platinum capsules. The analysis was performed in a TG-60 equipment (TA-60WS, Shimadzu, Kyoto, Japan), under a nitrogen atmosphere, with a flow rate of 50 mL/min, an initial temperature of 30°C, and a heating rate of 10°C/min until reaching the final temperature of 500°C. The OriginPro® 8.5 software was used to process the data.
2.2.11 Contact angle with water
The hydrophobicity of the electrospun fibers was evaluated by measuring the contact angle using an optical tensiometer (TL100, Biolin Scientific, Sweden). Initially, the fibers were collected for 60 min (with the parameters mentioned in Section 2.2.6) on glass slides to form a fiber membrane. Afterward, a drop of distilled water (7 µL) was placed on the sample, and the water absorption time was recorded in a digital camera for 60 s. The data obtained were processed in the OriginPro® 8.5 software to obtain a graph of the absorption behavior. The analysis was performed at room temperature (22 ± 2°C) (Ahmad et al., 2015).
2.2.12 Antioxidant activity
The in vitro antioxidant activity was determined by the ability to eliminate 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), and hydroxyl (OH) radicals according to the method of Vinholes et al. (2011), with adaptations. The electrospun fibers (5 mg) were dissolved in 1.5 mL of 75% ethanol (in distilled water, v/v), and from there, each sample was added to a 96-well microplate, and serial dilutions were performed. The maximum inhibition percentage of antioxidant activity against all the free radicals was calculated using Eq. 3, and assays were performed in triplicate.
Where: “Absblank” is the absorbance of the control (blank), and “Absample” is the absorbance of the sample.
2.2.12.1 2,2-Diphenyl-1-picrylhydrazyl
For the DPPH radical, aliquots of 25 µL (previously prepared according to Section 2.2.12) and 250 µL of a 0.6 mM DPPH solution were added. The plates were incubated in the dark for 30 min and subsequently analyzed in a microplate spectrophotometer (SpectraMax 190, Molecular Devices, USA) at a wavelength of 515 nm.
2.2.12.2 Nitric oxide
For the NO radical, 50 µL of the sample (previously prepared according to Section 2.2.12) was added to a 96-well plate, followed by 50 µL of sodium nitroprusside (20 mM). The mixture was incubated at 22°C under light for 60 min. Then, 50 µL of 2% phosphoric acid solution and 50 µL of Griess reagent were added. The microplate was incubated again for 10 min at 22°C in the dark and then read in a microplate spectrophotometer (SpectraMax 190, Molecular Devices, USA) at a wavelength of 562 nm.
2.2.12.3 Hydroxyl
For the hydroxyl radical, 25 µL aliquots (previously prepared according to Section 2.2.12) were added to a 96-well plate along with 110 µL of iron sulfate solution (8 mM), 50 µL of hydrogen peroxide solution (7.18 mM), and 74.2 µL salicylic acid solution (3 mM). The plates were shaken for 10 s and incubated for 30 min at 37°C inside a microplate spectrophotometer (SpectraMax 190, Molecular Devices, USA). Subsequently, the reading was performed at a wavelength of 515 nm.
2.3 Experimental design and statistical analysis
The experiment was carried out with biological and technical triplicates, each represented by a sample and treatments’ respective extraction and application. Data were submitted to analysis of variance (ANOVA), and Tukey’s test was applied to compare means with a statistical significance level of p ≤ 0.05.
3.1 Total phenolic, flavonoid, and anthocyanin content of ROE
Red onion is a source of phenolic compounds, which provides high antioxidant activity due to the high bioactive compound concentration that, for the most part, is present in the skin (outer part). However, most of these compounds are also found in the bulb (internal part), among which we can mention flavonoids, which are phenolic compounds responsible for conferring high antioxidant activity. In addition to flavonoids, red onions are rich in anthocyanins, a group of pigments belonging to the family of phenolic compounds responsible for eliminating free radicals, providing more excellent antioxidant activity (González-de-Peredo et al., 2021).
The ROE showed a total phenolic content of 1922.53 mg GAE/100g on a wet basis, which is higher than the data reported by Metrani et al. (2022), who evaluated two red onion cultivars and found values ranging from 140 to 157 mg GAE/100g of fresh sample and Salamatullah et al. (2021), who obtained values ranging from 64.35 to 815.93 mg GAE/100g on a dry basis in red onions subjected to different drying temperatures.
As for the total flavonoid content, the extract showed a content of 1897.62 mg QE/100g on a wet basis, a result also higher than Salamatullah et al. (2021), who obtained values ranging from 12 to 729.56 mg CA/100g on a dry basis when subjecting red onions to different drying temperatures. For the total anthocyanin content, the ROE showed a content of 11.36 mg C3GE/100g on a dry basis, corroborating other studies that also evaluated the ROE, including Oancea and Drághici (2013), who found values ranging from 0.12 to 7.93 mg/100g for different red onion cultivars. Metrani et al. (2020) also evaluated two red onion cultivars (‘Honeysuckle’ and ‘Sweet Italian’) and found total anthocyanin contents of 10.3 and 8.6 mg/100 g, respectively. Anthocyanins are responsible for exerting various biological effects; extracts rich in anthocyanins can prevent or reduce the risk of developing multiple pathologies, including obesity, cardiovascular disease, and the onset of cancer because they are antioxidant agents (Colina-Coca et al., 2017).
Differences in the total phenolic, flavonoid, and anthocyanin content may be related to the method used for extraction and identification, edaphoclimatic conditions, soil type, and management (Metrani et al., 2020; Salamatullah et al., 2021).
3.2 Volatile composition of ROE headspace by GC-MS (HS-SPME-GC-MS)
Volatile compounds contribute to the overall sensory attributes of food products and generally determine their acceptability and quality. For example, studies have been carried out in onions on the relationship between volatile compounds and sensory characteristics of bulbs, showing the importance of volatile composition for sensory attributes (Cozzolino et al., 2021; Lekshmi et al., 2014). Nine VOCs were detected and identified in the ROE sample (Supplementary Material Table S1). The heat map (Fig. 1) illustrates the quantification of identified volatile organic compounds belonging to the classes of ketones, hydrocarbons, aromatics, alcohol, and acids, among others, which are determined by the color of the rectangle, where, in the color scale, the highest concentration was represented by the color red.
The major compound was 3(2H)-furanone, 2-hexyl-5-methyl (~ 0.04 mg/mL), which belongs to the furanone class. This compound has already been reported in onion skin extract, such as by Constantin et al. (2021). This compound is considered a product of the rearrangement of sulfur; it has also been previously reported in onion ‘shallot’ and is attributed as a fatty acid breakdown product derived from the onion tear factor (Tocmo et al., 2014). The second volatile compound was squalene (~ 0.03 mg/mL), a natural 30-carbon triterpene found in the hydrolipidic profile of some plants, as reported in the literature for a volatile profile of onion (Lekshmi et al., 2014). Squalene is synthesized in all cell types because it is a critical intermediate in forming eukaryotic sterols and bacterial hopanoids.
For this reason, various plants have become valuable sources for squalene isolation, as it is widely present in nature but in substantial amounts. The third major compound, o-cymene (~ 0.02 mg/mL), is an aromatic hydrocarbon that has already been reported in essential oils extracted from different matrices, such as from plants of the genus Allium (Pande et al., 2015). The compounds 2-octanone and 2-decanone were also identified (~ 0.01 mg/mL). In black garlic (Allium sativum L.), the compound 2-octanone has already been identified by Najman et al. (2022), who reported that this volatile could be detected due to aging. The authors attributed that this substance can contribute to the roasted, burnt, caramelized, green, and fruity aroma. There are reports of the presence of eugenol (~ 0.01 mg/mL), in plants of the genus Allium, as in the essential oil of garlic (Allium sativum L.) (Dziri et al., 2014).
The eugenol identified in the extract (~ 0.01 mg/mL) is a phenylpropanoid, a phenolic aromatic compound, weakly acidic and slightly soluble in water and organic solvents. The terpinen-4-ol (~ 0.01 mg/mL) is a monomeric compound in many essential oils. Hexadecanoic acid (~ 0.01 mg/mL), which is a saturated fatty acid widely reported to be present in onions as well as onion extracts and onion skin extracts (Acharya et al., 2019; Constantin et al., 2021). Finally, the last volatile organic compound identified was 2,4,4-trimethyl-1,3-pentanediol 1-isobutyrate (~ 0.01 mg/mL), an alcoholic ester.
The characteristic odors and flavors and the biological activities of the onion are related to its composition of VOCs. Disulfides are the most abundant class of VOCs, followed by trisulfides (Cecchi et al., 2020; Cozzolino et al., 2021; Lekshmi et al., 2014). Nonetheless, these two classes were not observed in the ROE volatile analysis in this study, suggesting that the extract production process may affect the presence of these compounds. The studies found in the literature show a variable composition of VOCs referring to onions, mainly red. This is directly attributed to the parts of the onion, its origin, cultivar, species, and the edaphoclimatic conditions under which they are cultivated.
3.3 Apparent viscosity and electrical conductivity of polymer solutions
The polymeric solutions used in producing fibers with different concentrations of ROE were evaluated for apparent viscosity and electrical conductivity (Table 1) since these parameters influence the electrospinning behavior and the quality of the fibers produced (García-Moreno et al., 2018). A gradual increase in electrical conductivity can be observed when the amount of ROE added to zein polymeric solutions increases from 10 to 40% (v/w), associated with increased ionizable groups in the solutions. The same behavior was observed by Prietto et al. (2018), who added anthocyanins extracted from red cabbage to zein polymeric solutions to obtain a pH-sensitive indicator produced by electrospinning.
An inverse behavior was observed for apparent viscosity compared to electrical conductivity since as the concentration of ROE in polymeric solutions increased, there was a decrease in apparent viscosity (Table 1). Nevertheless, studies show the opposite behavior when incorporating a jambolan extract (Santos et al., 2022) and red cabbage extract (Prietto et al., 2018), rich in anthocyanins, in zein polymeric solutions. This difference may be related to the use of a liquid extract in the present, while the aforementioned authors used a dry extract. Thus, adding ROE increased the presence of liquids in the zein polymeric solution, decreasing viscosity as it was added and increasing its concentration. Polymer solutions with low viscosity tend to form fibers with beads along their length, as seen in our study (especially in fibers with 40% ROSE).
3.4 Loading capacity
The LC is a crucial parameter to quantify the concentration effect of the compounds loaded on the polymeric matrix. Some factors can influence this process, such as the polymer concentration and the bioactive compounds in the polymeric solution, which significantly increase or reduce loading.
The ROE concentration added to the zein electrospun fibers affected the LC (p < 0.05), according to the increase in ROE concentration, there was a reduction in LC from 91.5 to 77.3% (Table 1). We observed no significant difference (p < 0.05) between fibers with 10 and 20% ROE, with a drop in LC for fibers with 30 and 40% ROE, which also did not show a significant difference between each other.
Regarding fibers with 10% ROE, there was a loss of approximately 14.7 to 15.5% for fibers with 30 and 40% ROE in the LC, respectively. This behavior of decreasing LC as ROE was added may be related to the saturation of the active sites available in the polymer used (zein) due to the progressive increase of ROE in polymeric solutions or even a lower interaction of the material wall thickness and ROE or even losses during the electrospinning process.
In order to be considered an effective process, minimum values of 70% loading of the compounds are suggested so that the process becomes industrially viable (Cruz et al., 2022). The LC values reported in this study showed that the polymer used as wall material is highly effective for loading bioactive compounds. Furthermore, encapsulation aims to protect the compounds against external environmental factors, such as humidity, light, and oxygen, so that there is no loss of their bioactivity and functionality (Cruz et al., 2021). This can promote desirable characteristics in active packaging or a food matrix. In addition, encapsulation prevents the vital sensory attributes of onion from being transferred to the food product when undesirable.
Santos et al. (2022) evaluated the loading capacity of ultrafine fibers produced using zein as a wall material to encapsulate jambolan extract at concentrations of 20, 30, and 40% (w/w). The authors obtained values ranging from 58.0 to 66.8% of loading capacity. Cruz et al. (2023) reported that the loading capacity was 67 to 78% for ultrafine fibers based on sweet potato starch incorporated with 3, 6, and 9% of red onion skin extract. These differences found for the loading capacity may be related to the plant material analyzed or even to the method and parameters used to encapsulate the compounds.
3.5 Morphology and diameter of zein fibers
In order to evaluate the effects of incorporating different concentrations of ROE (0, 10, 20, 30, and 40%, v/v) on zein electrospun fibers production, the morphological characterization and size distribution of the materials obtained were carried out (Fig. 2). The micrographs show that the conditions used in the processing guaranteed to obtain random and continuous fibers. Furthermore, the incorporation of ROE, especially at the highest concentration (40%, v/v), produced fibers with the presence of beads, which did not occur for the fibers without ROE incorporation.
Nevertheless, the increased incorporation of ROE in the zein electrospun fibers ensured a reduction in diameter and greater homogeneity of the materials since the variation in diameters also decreased as the concentration of ROE ranged from 0% (512 ± 132 nm) to 40% (224 ± 50 nm). Thus, it was possible to obtain a reduction of 56.25% in the average diameter of the fibers by incorporating ROE at the highest concentration. According to Neo et al. (2013), polymeric solutions with higher viscosity generate greater molecular entanglement and, thus, increase the fibers’ diameter. The same trend was observed in our study, as the greater the viscosity of the polymeric solutions, the greater the diameter obtained.
Furthermore, as the electrical conductivity of the polymeric solutions increased (with the increase in ROE concentration), the fibers diameter decreased. This behavior is justified by a lower stretching of the solution jet, resulting in higher viscosity and lower electrical conductivity of the fibers-forming solutions, which, when combined, result in fibers with larger diameters.
3.6 Fourier transform infrared spectroscopy
The FTIR analysis was used to characterize the chemical nature of the electrospun fibers and their precursor materials (Fig. 3). A broad band centered at 3433 cm− 1 was observed for the unencapsulated ROE, characteristic of N-H stretching of proteins and O-H bond stretching of carbohydrates and water, whereas the band 2921 cm− 1 is characteristic of C-H stretching of aliphatic groups (Chen et al., 2020; Santos et al., 2022). The band around 1635 cm− 1 is attributed to the C-C stretch of the phenyl ring that, in Allium plants, is present in high levels as polyphenolic constituents (Lu et al., 2011). Additionally, at 1105 cm− 1, a “shoulder” peak is observed, which is characteristic of the carbohydrates in onions (Lu et al., 2011). Furthermore, in the range of 1000–950 cm− 1, the bands are attributed to the elongation of the C = C and C-OH bonds located in the aromatic rings of anthocyanins are observed (Santos et al., 2022).
Regarding the spectra obtained for the zein polymer and fibers 0% ROE, no alterations were observed (Fig. 3); both showed broad bands around 3500–3000 cm− 1, which are attributed to the elongation of the O-H and N-H bonds of the hydroxyl and amide groups present in zein. In the 3000 to 2850 cm− 1 range, bands are observed and refer to the stretching of C-H bonds and aliphatic CHX groups. In the range of 1648–1640 cm− 1, bands are observed, referring to the stretching of C = O of the major amide (Li et al., 2020). Finally, bands around 1535 and 1449 cm− 1 are observed, which refer to the elongation of C = O and C-N bonds, respectively (Neo et al., 2013), suggesting the electrospinning process did not affect the structure of the protein. However, it interrupted intramolecular bonds such as hydrogen bonds. Minor changes were observed in the FTIR spectra for fibers containing different concentrations of encapsulated ROE (10, 20, 30, and 40%) compared with the spectrum obtained for zein. In these spectra, a slight narrowing of the band centered around 3500 cm− 1 is observed and attributed to the elongation of the O-H and N-H bonds compared to pure zein. According to Santos et al. (2022), this narrowing may be related to intermolecular interactions (hydrogen bonds) between the polymer (zein) and anthocyanins present in ROE. In addition, the fibers containing different concentrations of ROE showed characteristic bands of the anthocyanin aromatic rings (bands around 1000–950 cm− 1), which are more evident at higher ROE concentrations in the fibers due to hydrogen bond formation between zein and ROE, thereby confirming the interaction between the polymer and ROE and its effective encapsulation.
3.7 Thermogravimetric analysis
Thermogravimetric analysis of zein electrospun fibers loaded with different concentrations of ROE and its constituents (pure ROE and pure zein) allowed us to investigate the mass loss of each sample as a function of temperature and the influence of the electrospinning process on these losses (Fig. 4). The ROE (unencapsulated) showed two phenomena of mass loss based on TGA and DTG curves (Figs. 4a and 4b). Within a greater mass degradation, the first event occurred in the approximate range of ~ 33 to 167 ºC, with a peak temperature at ~ 140 ºC. With the less mass loss, the second event occurred at a degradation range of roughly ~ 180 to 225°C and a peak temperature of ~ 204°C. These mass loss phenomena are probably related to the evaporation of water, volatile compounds, and the degradation of molecules sensitive to this temperature range.
During thermal analysis, the pure zein and electrospun fibers with 0 to 40% ROE showed similar behavior. These samples presented the first mass loss at peak temperature at ~ 50 ºC, which probably refers to the loss of water and volatile compounds in the samples. A second thermal event was observed around ~ 170°C and the third event, responsible for the greatest mass loss of pure zein and fibers, which occurred in the range of ~ 195 to ~ 400 ºC (Fig. 4a), as demonstrated by the highest rates in the DTG diagram (Fig. 4b). These events possibly refer to breaks in the polymer chains due to the cleavage of bonds and biopolymer degradation (Altan et al., 2018; Antunes et al., 2017; Neo et al., 2013). The electrospinning process could not significantly change the polymer (zein) behavior in the rate of mass loss as a function of temperature, given the similarity observed between the curves of pure zein and fibers 0% ROE (i.e., electrospun fibers containing only zein). Moreover, the process of encapsulation of ROE in zein fibers could improve the thermal stability of the extract due to an increase in degradation temperatures of encapsulated ROE into zein fibers (degradation range of ~ 195 to ~ 400 ºC) compared to unencapsulated ROE (degradation range of ~ 33 to ~ 167 ºC) (Fig. 4).
In addition, the similarities between the TGA and DTG curves of zein fibers incorporated with 10 to 40% ROE compared to pure zein fibers (fibers 0% REO) indicate efficient extract loading into the wall material, corroborating the findings for LC presented in Section 3.4. Similar behavior was observed by Altan et al. (2018) and Evangelho et al. (2019), who incorporated active compounds such as carvacrol and folic acid into zein ultrafine fibers, respectively.
3.8 Contact angle with water
The hydrophilicity of zein membranes with ROE was measured by contact angle with water (Fig. 5). At the initial moment of 2 s, after the drop fell, the zein fibers membranes showed lower values for contact angle as ROE was incorporated. After 60 s, the membranes continued to decrease in the contact angle, which was smaller as the concentration of ROE in the fibers increased (similar behavior to the data for 2 s of analysis) except for the fibers with 20 and 30% ROE, which showed no significant difference (p > 0.05). Possibly, the highest mean contact angle values for zein fiber membranes without the addition of ROE occurred due to the presence of hydrophobic amino acid residues in the protein, which can reduce its interaction with water and, consequently, make the membrane more hydrophobic or less hydrophilic (Prietto et al., 2018).
As for the fibers membranes with ROE, which showed a reduction in contact angles as ROE was added, it is likely due to the incorporation of anthocyanin molecules, which are highly hydrophilic and water-soluble compounds, increasing the interaction of the membranes with the drop of water and making them more hydrophilic (Santos et al., 2022). All the fibers in this study presented contact angles below 90°, indicating hydrophilic surfaces according to the classification reported by Ahmad et al. 2015.
The more excellent permeability of the fibers with the addition of ROE can be helpful in different applications, including in active packaging, allowing the compounds to interact with the food product in a controlled and specific way, providing greater preservation due to the antioxidant properties of ROE. Similar behavior was also observed by Santos et al. (2022) when evaluating the contact angle in zein fibers added with anthocyanins from jambolan (at concentrations of 20, 30, and 40%, w/w). The authors reported that the higher the anthocyanin concentrations, the greater the permeability of the fibers and, consequently, the lower the contact angle.
3.9 Antioxidant activities
The antioxidant activities of the unencapsulated ROE (Table 1) were more significant than its encapsulated form for all radicals evaluated (DPPH, NO, and OH). The lower antioxidant activity in electrospun fibers with ROE is possibly due to the non-complete release of compounds retained in the zein fibers. As for the DPPH radical, there was an increase in antioxidant activity with increasing ROE into zein fibers. This behavior helps to confirm ROE encapsulation and the effectiveness of the encapsulation technique.
Similar behavior was observed for the NO radical. However, no significant difference was observed for zein electrospun fibers added with 30 and 40% ROE. As for the OH radical, the fibers added with 20, 30, and 40% of ROE did not show significant differences. Pure zein electrospun fibers (i.e., 0% ROE) did not present antioxidant activity against the evaluated radicals.
Despite not being produced by the human body, the DPPH radical is widely used to determine antioxidant activity. It is one of the few stable radicals available on the market, capable of accepting an electron or hydrogen radical to become a stable molecule. The reaction for determining the antioxidant capacity of compounds against DPPH occurs when its radical form is reduced by an antioxidant (Radünz et al., 2021), in this case, ROE bioactive compounds. Nevertheless, reactive oxygen species (ROS) are constantly produced in the human body through oxidative metabolic processes, not showing toxic effects at low concentrations; NO and OH radicals stand out among the ROS. The OH is the most abundant and destructive species, acting on lipid peroxidation and promoting mutations at the DNA level by modifying the purine and pyrimidine bases. However, NO can act in lipid peroxidation, promoting the release of pro-apoptotic factors and cell death. In addition, they are capable of causing food product deterioration and are also related to several diseases in living organisms (Charrier & Anastasio, 2011; Radünz et al., 2021).
Although zein electrospun fibers added from ROE have lower antioxidant activities than unencapsulated ROE; it is essential to mention that NO and OH radicals are important molecules for maintaining the proper functioning of living organisms, and their complete inhibition would be harmful. Thus, lower rates of capturing these radicals are desirable and possibly sufficient to reduce the concentration of excess oxidant species and, consequently, control oxidative stress, protecting cells from damage that could be caused by these radicals (Radünz et al., 2021). In addition, some studies have demonstrated the antioxidant activity of onions and onion extracts, such as (Prietto et al., 2018; Colina-Coca et al., 2017; Zhao et al., 2021).
Natural extracts are sources of antioxidants, including bioactive compounds, whose activity is widely explored in the pharmaceutical, cosmetic, and food industries and can replace the synthetic antioxidants commonly used in the food and packaging industry. In addition, research reports that the antioxidant activity of volatile compounds is directly related to the total content of phenolic compounds in this type of sample (volatile oils and organic extracts). Thus, in this study, our findings showed that zein electrospun fibers added with different encapsulated ROE concentrations showed potential as natural antioxidants due to sufficient free radical scavenging capabilities, which can control oxidative processes in some foods, for example, with a short shelf life.
The red onion extract (ROE) showed high total phenolic, flavonoid, and anthocyanin content. The volatile organic compounds found in major proportions were 3(2H)-furanone, 2-hexyl-5-methyl (~ 0.04 mg/mL), and squalene (~ 0.03 mg/mL). The polymeric solutions showed a trend in which, as ROE (0, 10, 20, 30, and 40%, v/v) was incorporated, there was a reduction in apparent viscosity and increased electrical conductivity. The ROE loading capacity into zein electrospun fibers was high and ranged from 77.3 to 91.5%, and the ROE encapsulation improved thermal stability.
The electrospun fibers presented random and continuous morphology, and there was a reduction in size distribution at higher ROE concentrations, ranging from 224 to 512 nm. The increased ROE concentrations in zein electrospun fibers also reduced the contact angle of the membranes, with hydrophilic characteristics. The antioxidant activities of the unencapsulated ROE were 67.3, 74.5, and 57.7% against DPPH, NO, and OH radicals, respectively. The antioxidant activity of the electrospun fibers ranged from 32.5 to 57.3% against the different free radicals evaluated. Lastly, zein electrospun fibers with encapsulated ROE showed bioactive potential for applications in different food products and active food packaging.
Acknowledgments
The Southern Electronic Microscopy Center (CEME-SUL) of the Federal University of Rio Grande (FURG), and the Center for Development and Control of Biomaterials (CDC-Bio) of the Federal University of Pelotas (UFPel). We would also like to thank Atlas Assessoria Linguística for language editing.
Author Contribution
Elder Pacheco da Cruz: Conceptualization, Methodology, Software, Formal analysis, Data curation, Investigation, Writing–original draft, Writing–review & editing.Juliani Buchveitz Pires: Formal analysis, Data curation, Investigation, Writing–original draft, Writing–review & editing. Estefani Tavares Jansen: Formal analysis, Data curation, Investigation, Writing–original draft, Writing–review & editing. Felipe Nardo dos Santos: Formal analysis, Data curation, Investigation, Writing–original draft, Writing–review & editing. Laura Martins Fonseca: Conceptualization, Writing–original draft, Writing–review & editing. Helen Cristina dos Santos Hackbart: Methodology. Marjana Radünz: Methodology. Elessandra da Rosa Zavareze: Conceptualization, Supervision, Validation, Funding acquisition, Writing–review & editing. Alvaro Renato Guerra Dias: Conceptualization, Supervision, Validation, Funding acquisition, Writing–review & editing.
Funding
The authors would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES (Finance Code 001), Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPQ, and Fundação de Amparo a Pesquisa do Estado do Rio Grande do Sul – FAPERGS for funding.
Data Availability
The data used to support the findings of this study are included in the article. Conflict of Interest The authors declare no competing interests.
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No competing interests reported.