Electrical conductivity and apparent viscosity of polymeric solutions
The polymeric solutions were initially characterized in terms of electrical conductivity and apparent viscosity. The addition of jambolan extract (20, 30, and 40%) reduced the electrical conductivity of zein polymer solutions (Table 1), but this reduction did not compromise the fiber formation process. This behavior was expected, considering that these variables are affected, among other factors, by the polymer concentration [25].
Table 1
Electrical conductivity and apparent viscosity of the polymeric solutions formed by zein and different concentrations of jambolan extract and encapsulation efficiency (EE) of jambolan extract in zein fibers.
Jambolan extract
(%; w/v)
|
Electrical conductivity
(µS/cm)
|
Apparent viscosity (cP/cm)
|
EE (%)
|
0
|
1821.00 ± 1.00a
|
126.60 ± 0.17d
|
-
|
20
|
1561.00 ± 2.65b
|
154.70 ± 0.17c
|
66.83 ± 1.07a
|
30
|
1463.00 ± 3.61c
|
171.10 ± 0.62b
|
62.65 ± 0.64b
|
40
|
1347.67 ± 1.51d
|
204.47 ± 0.65a
|
58.04 ± 0.32c
|
*Values expressed as mean ± standard deviation (n = 3). Different letters in the same column differ from each other (p < 0.05).
The addition of jambolan extract to the zein polymeric solution increased the apparent viscosity of the solutions (Table 1), probably due to the interaction of the extract and the protein, with the formation of intermolecular hydrogen bonds between them. According to Aytac et al. [26], the increase in apparent viscosity is caused by the greater entanglement of polymer chains in the solution. Similar behavior was observed by Evangelho et al. [17] when evaluating the electrical conductivity and apparent viscosity in solutions zein incorporated with folic acid. In this study, the authors reported that as the concentration of folic acid increased, the electrical conductivity decreased and there was an increase in the apparent viscosity of the polymeric solutions. Considering that the electrical conductivity and apparent viscosity obtained were within the expected range, the characterization was continued by analyzing the morphology and size distribution.
Morphology and size distribution
The parameters used in the electrospinning process allowed the formation of ultrafine fibers, as visualized by SEM. Overall, the fibers showed to be homogeneous and continuous, without the presence of beads (Figure 1). The incorporation of different amounts of jambolan extract (20, 30, and 40% w/w) did not affect the morphology of the fibers obtained. However, the average diameter of the fibers changed, which can be attributed to the increase in the apparent viscosity of the solutions when the extract concentration was increased (Table 1). In more detail, the mean diameter ranged from 472 to 622 nm, with the largest diameter observed for the fiber incorporated with 30% (w/w) of jambolan extract (Figures 1e-f).
In general, differences in fiber diameters obtained by electrospinning can be attributed to different factors, including the conditions of the electrospinning process, such as feed rate, voltage, and collector distance, and characteristics of the electrospinning solution, such as apparent viscosity, conductivity electrical and surface tension [25]. In our study, all samples were prepared in the same process conditions. Therefore, the characteristics of the solution (electrical conductivity and apparent viscosity) were those that exerted influence on the average diameter of the fibers. The increase in the concentration of jambolan extract imparted a higher viscosity to the polymeric solutions, which may have influenced the formation of fibers with larger diameters. For example, the fibers produced without jambolan extract had an average diameter of 472 nm, those with 20%, a diameter of 596 nm, those with 30%, a diameter of 622 nm, and those with 40%, a diameter of 562 nm.
Studies carried out with ultrafine fibers of zein added with different concentrations of extracts of jabuticaba and red cabbage showed diameters ranging from 583 to 572 nm and 444 to 510 nm, respectively [6,27]. Evangelho et al. [17] reported an average diameter ranging from 369 to 702 nm for fibers produced with zein (30% w/v) and folic acid (0, 0.5, 1, and 1.5%). Altan et al. [28] when producing fibers using 30% (w/v) zein solutions, reported an average diameter of 604 nm, while incorporated zein fibers of 5, 10, and 20% (w/v) of carvacrol had diameters of 647, 539 and 553 nm, respectively. According to these results, it can be affirmed that, in this study, the concentration of extract added to the polymeric solutions had a strong effect on the formation of fibers, presenting an increase in the average diameter.
Encapsulation efficiency (EE)
The EE was estimated from the quantification of the total extract that was kept encapsulated in zein fiber, determining the concentration of analytes by HPLC. Fibers prepared with 20% jambolan extract showed the highest EE (Table 1), while fibers prepared with 40% jambolan extract showed the lowest EE, which reveals an inverse relationship between the concentration of the compound and its EE in the zein fibers. The reduction of the values computed for EE with the increase of the concentration of jambolan extract may be related to an excess of compounds present in the solution, which makes it difficult to incorporate them into the protein matrix, due to the reduction of the active sites of the protein. Similar behavior was also observed by Evangelho et al. [17], that reported a reduction in EE as the concentration of folic acid in zein fibers increased.
Fourier Transform Infrared Spectroscopy (FTIR)
The FTIR technique was used to characterize the chemical nature of the fibers and their precursor materials (Figure 2a). The FTIR spectrum obtained for the jambolan extract showed a broad band centered at 3416 cm-1 referring to the stretching of the O-H bond of the hydroxyl groups of anthocyanins. Bands were also identified at 2934 cm-1, which are related to the C-H stretching of the aliphatic groups [29]. In the wavenumber regions of 1742 and 1620 cm-1, bands were noticed due to the asymmetric and symmetric stretching of the C=O bond of carbonyl groups [30]. Also, in the range of 1100–950 cm-1, bands attributed to the stretching of the C=C and C-OH bonds present in the aromatic rings of the anthocyanins were observed [31].
Comparatively, the spectra of zein and pure zein fiber showed no significant change, as observed in Figure 2a. In both spectra, broad bands were observed in the region between 3500–3000 cm-1 referring to the stretching of the O-H and N-H bonds due to the hydroxyl and amide groups present in the zein structure [32]. In the range of 2900–2790 cm-1, bands due to the stretching of the C-H bond of the aliphatic CHx groups were observed. The band at 1646 cm-1 is attributed to the C=O stretching of the primary amide, while the band at 1537 cm-1 is attributed to the C=O stretching of the secondary amide [30]. Finally, the characteristic band associated with the stretching of the C-N bond was observed at 1446 cm-1 [31]. The observation of these bands suggests that the electrospinning process did not change the chemical structure of the protein; however, it interrupted the intramolecular interactions between them, such as hydrogen bonds, for example.
The FTIR spectra obtained from fibers containing different concentrations of jambolan extract (20, 30, and 40%), exhibited bands assigned to the functional groups of zein with minor modifications. In these spectra, it was observed a narrowing in the band assigned to the stretching of the O-H and N-H bonds (centered around 3500 cm-1) in comparison with the spectrum of pure zein. This is probably due to the intermolecular interactions between zein and the anthocyanins present in the jambolan extract by hydrogen bonding [33]. Moreover, it was also observed an overlap of the C=O bond bands from jambolan by the amide bands (primary and secondary) present in zein. Comparing these spectra, a slight increase in the shoulder-type band around 1740 cm-1 (C=O stretch) is observed as a function of the increase in the amount of jambolan extract encapsulated. In general lines, these finds confirm the presence of the extract in the fibers. Additionally , the spectra obtained for the fibers containing jambolan extract also showed the characteristic bands of the anthocyanins aromatic rings (range of 1100–950 cm-1), which are more evident with increasing extract concentration. Finally, as there was an increase in the amount of jambolan extract incorporated into the fibers, a widening in the band related to the stretching of the O-H and N-H bonds (mainly towards the right) was observed. This is probably due to the interaction between zein and the extract via hydrogen bonds, corroborating to the increase in solution viscosity as the extract concentration increased.
Differential Scanning Calorimetry (DSC)
The DSC technique was used to evaluate the thermal behavior of the pure materials and the prepared fibers. The DSC curves obtained are shown in Figure 2b, respectively. The DSC curve of pure zein showed an endothermic peak in the range of 30–120 °C with a maximum of 50 °C, corresponding to the water loss [29]. A smaller endothermic peak (centered at 147 ºC) can be associated with the breaking of hydrogen bonds and other molecular interactions present in the zein structure [34]. On the other hand, the DSC curve of pure zein fiber showed an endothermic peak in the temperature range between 30–125 °C, with a maximum of 75 °C, suggesting that the electrospun material has greater ordering compared to pure zein. Due to this greater ordering, that is, greater organization of the zein structure after the electrospinning process, there is a need for greater energy for the thermal event to occur, which consequently increases the enthalpy and thermal stability of the material [35]. There is also a slight reduction in the endothermic peak at 147 °C, probably due to changes caused in the zein structure as a result of the electrospinning process. Such changes were also observed by the FTIR analyses.
The DSC curve of the jambolan extract showed three endothermic peaks. The first, with a maximum at 80 °C, corresponding to the loss of water and volatile compounds present in the extract. The maximum temperatures for the second and third peaks were registered at 147 and 193 °C, respectively. These last two peaks are attributed the structural thermal degradation of the anthocyanins present in the extract as described in the literature [36]. The DSC curves of the fibers containing the jambolan extract (20, 30, and 40%) presented two endothermic peaks. The first peak in the temperature range of 30–120 °C likely due to the removal of water from the fibers. The second peak was observed between 100–200 °C, which is characteristic of protein denaturation [37]. Increasing the concentration of jambolan extract increased the area of the first endothermic peak, which in turn increased the enthalpy difference of the material. This fact is probably related to the greater hydrophilicity of the material, making the water removal process require more energy [36]. A decrease in the temperature at which the event occurred (from 128 to 102 ºC) was also observed. Probably this occurs due to the interactions of jambolan extract with zein, justified by the possible intermolecular interactions between the polymer and the compounds present in the extract [31]. These results corroborate with the data obtained from the viscosity and FTIR analysis.
Thermogravimetric analysis (TGA)
The TG/DTG curves obtained for the pure compounds and for the ultrafine fibers containing the jambolan extract are shown in Figure 3. The TGA curve of jambolan extract showed a thermal profile with three stages of mass loss analyzed temperature range. The first, with a maximum of at around 67 °C (about 10% of mass loss) can be attributed to the loss of water from the sample. The second initiate at 150 °C (resulting in 27% mass loss) reached a maximum at 199 °C and is attributed to the degradation of the lateral hydroxyl groups present in the anthocyanin structure. The third stage occurred between 250–450 °C with a maximum at 315 °C and corresponds to the thermal degradation of the compound (45% mass loss) [38]. The TGA curves obtained for pure zein and zein fibers showed similar thermal profiles, with only two stages of mass loss. The first stage between 30–100 °C (about 10% mass loss) is due to water loss and the second stage (about 70% mass loss), which occurs between 250–350 °C is characteristic of thermal degradation of proteins [30].
The TG/DTG curves of the fibers containing the jambolan extract showed three stages of mass loss. The first, with a maximum of around 67 °C (about 10% of mass loss) is related to water loss. The second stage between 100–200 °C with a maximum at 199 °C (about 10% mass loss) is associated with the degradation of the lateral hydroxyl groups of the structure of the anthocyanins present in the extract. The third and final stage between 250–400 °C (about 40% mass loss) corresponds to the thermal degradation of the protein [32]. Compared with the fibers without the extract, there is an increase in the DTG peak with a maximum at 199 ºC as the concentration of extract in the fibers increases. In contrast, the DTG peak related to the thermal degradation of zein (at 315 ºC) reduces as the extract concentration increases. As demonstrated by the viscosity, FTIR, and DSC analyses, there is an interaction between the extract compounds and the zein structure, which probably interferes with the thermal degradation process of this protein. However, as there is no change in the temperature range at which this thermal event occurs, we can suggest that the incorporation of the extract, even at concentrations of up to 40% w/w, does not alter the stability of the fibers.
Fiber contact angle
The contact angles of the ultrafine fibers with different concentrations of jambolan extract are shown in Figure 4. The pure zein fibers (Figures 4a and 4b) presented an initial contact angle of 94.4° in the immediate instant of droplet fall, being reduced to 26.4° after 3 seconds of droplet contact with the fiber surface.
The contact angle of the fibers decreased as the concentration of jambolan extract increased, from 89.0° (20% extract) to 64.4° (40% extract) and after 3 seconds of contact, it reduced from 20.7° (20% extract) to 14.5° (40% extract). This decrease in the contact angle is due to the hydrophilicity of anthocyanins present in the extract, implying highly hydrophilic fibers. Higher values for the contact angle of zein fibers are due to the presence of hydrophobic amino acids in the protein [39]. Similar behavior was also observed by Prietto et al. [6] that evaluating the contact angle in zein fibers added with anthocyanins from red cabbage. These authors reported that the higher the concentration of anthocyanins, the greater the wettability of the fiber. The greater wettability of zein fibers containing jambolan extract may be useful for applications in smart packaging, allowing the anthocyanins present in the fibers to interact quickly, emitting a color change response as a function of the change in the pH of the food during its storage.
Antioxidant activity
The incorporation of agents with antioxidant properties is extremely important when you want to produce food packaging. One of the main factors responsible for the intensification of food spoilage by microorganisms is oxidative degradation, in this sense, the use of natural antioxidants in food packaging can be an efficient alternative, capable of improving stability in food products sensitive to oxidation [40,41]. The results obtained for the antioxidant analysis of fibers containing different concentrations of extract (0, 20, 30, and 40% w/w) are shown in Figure 5.
The not encapsulated jambolan extract showed a scavenging capacity of 72% for the DPPH radical and 64% for the ABTS•+ radical (Figure 6). The high antioxidant activity of the extract is directly related to the phenolic compounds present, especially the anthocyanins, in a previous study carried out by Santos et al. [20], it was found that the main anthocyanin present in the jambolan extract was delphinidin, corresponding to 39.1% of the total anthocyanins identified. According to Nascimento-Silva et al. [42], delphinidin is the anthocyanin that is present in greater amounts in jambolan fruits, being responsible for the high antioxidant activity.
Zein fibers containing different concentrations of jambolan extract showed inhibition values of up to 40.5% for the DPPH radical (fiber containing 40% of extract). The highest concentrations of extract added (30 and 40%) were responsible for the highest percentages of inhibition (~39%), with no significant difference (p > 0.05) between them, for the inhibition of the DPPH radical. The decrease in the antioxidant activity of the encapsulated extract in relation to the pure one may be due to the degradation of the compounds that may occur during the electrospinning process; external factors (light, temperature, and oxygen) are usually responsible for this decrease.
For the ABTS•+ radical, the antioxidant capacity of the fibers was higher when compared to the DPPH radical. Furthermore, a gradual increase in the scavenging activity of this radical was observed as the concentration of jambolan extract present in the fibers increases. Herein, the highest antioxidant capacity was computed for the fiber containing 40% of the extract, with an average value of 58.8 % inhibition.
The greater inhibition activity of the ABTS•+ radical, compared to the DPPH radical, occurs due to a greater sensitivity of this radical for the identification of antioxidant activities, since it has faster reaction kinetics and also a greater response to antioxidants [43]. The encapsulation, by electrospinning, of jambolan extract in zein fibers is a highly promising alternative to increase the stability of the anthocyanins present, in addition to guaranteeing the protection of their functional properties, such as antioxidant activity. The incorporated fibers of jambolan anthocyanic extract produced by electrospinning may be a promising alternative in the development of active packaging due to its antioxidant activity, which can prevent or delay lipid oxidation in foods.