Pretreatment of the fiber material is really important to obtain good quality fibers. Dried and sieved rice bran should be defatted to remove the fat content (17%) [25] which could lead to the formation of emulsions during the electrospinning process. Emulsion formation can affect the physical parameters of the polymer solution as well as the fiber morphology. The defatted rice bran is then subjected to an alkali treatment to extract soluble dietary fibers (SDF) (pectin, proteins, carbohydrate and some amount of cellulose) [19]. The solid content of the supernatant was 30%.
After the extraction of SDF, the condition optimization for the electrospinning was done in order to avoid the formation of film and beads-on-string structures. Electrospun fibers with these artifacts are usually considered as “poor” quality fibers since they can reduce the total surface area significantly. As a result, these structures can directly reduce the enzyme loading capacity of the fibers thus leading to poor industrial sustainability.
Condition optimization
Solution pH
High conductivity or charge density results in more uniform fibers with fewer beads-on-string structures [26]. However, in this study, both extremes of pH values are not desirable since the enzyme undergoes denaturation at high pH values while the PVA molecules used as the viscosity modifier can undergo protonation at lower pH values leading for the formation of beads-on-string structures instead of uniform NF [26]. The optimum solution pH for electrospinning was set at 9.
Solution viscosity
Solution with 6% (w/v) PVA results in droplets and beads-on-structures rather than drawing into fibers. Smooth fibers with a very small amount of beads resulted when electrospinning was done with the dietary fiber supernatant amended with 8% (w/v) PVA. The solution viscosity and conductivity were optimized as 800 Cp, and 17.0 mS/cm, respectively.
Applied voltage
Applied voltage is another important parameter in determining the fiber morphology. The mechanism of charge transfer from the tip/needle to the collector through the flow of polymer is due to the voltage difference between two electrodes. Therefore, an increase in electrospinning current coincides with the mass flow rate away from the tip [25] as the applied voltage is proportional to the fiber diameter. In this study, the best quality nanosized fibers were obtained at a minimum applied voltage of 27 kV.
The distance between two electrodes
The distance between the needle and the collector is another parameter that determines the morphology and the diameter of the electrospun fibers. The distance has to be maintained in such a way that the fibers would get sufficient time to dry, before reaching the collector, by evaporation of the solvent used in fiber solution. Distances that are too close produced bead-on-fiber structures. When the distance was too long, the fibers started to gather at the edges of the collector. Droplet formation was observed at 10 cm distance, while the very small amount of fibers were formed at the edge of the collector at 20 cm. At 15 cm, a reasonable amount of fibers with nanoscale diameters were collected at the electrode. Hence, the distance between the needle and the collector was set as 15 cm.
Morphology of synthesized NF and CSNFP
The dietary fiber solution was successfully spun into NF and obtained a network structure with fiber diameter between 30 and 50 nm (Fig. 1a).
The fiber surface was smooth and a minimum number of beads-on-string structures were observed. These fibers were less than half the size of those reported, previously [19]. Interestingly, less morphological changes can be seen after surface modification of NF with phytase enzyme in the presence of STTP. However, a large number of beads of 400 nm diameter have been observed on the fibers of CSNFP composites. This network structure can offer an extra stability to the enzyme since it gives a better protection from external conditions. Also, the successive addition of negatively charged STTP might lead to the formation of crosslinks with positively charged ammonium groups in CSNFP. Moreover, NF might offer a high surface area for efficient encapsulation of the enzyme.
Thermal properties of synthesized NF
Differential scanning calorimetry (DSC) thermogram of the synthesized NF basically showed three main endotherms (Fig. 2).
The characteristic pattern is very similar to those reported by Zhang et al. [27] on PVA-soy blend NF and Fung et al. [19] on agro waste such as soya bean solid wastes, oil palm trunk and oil palm frond based NF. The first endotherm centered at 110 °C corresponds to the melting of the total SDF and other organic components present in the composite. The second endotherm centered at 215 °C can be attributable to the melting of PVA component, while the endotherm at 323 °C is related to subsequent decomposition of the composite under nitrogen environment.
First order differential thermal analysis (DTA) thermogram of NF is given in Fig. 3 compared to both individual PVA and rice bran.
For NF, weight losses were observed in three different temperature ranges (i) 39–99 °C, accounting for the removal of physisorbed water, (ii) 100–312 °C due to the loss of acetyl groups by transforming to acetic acid molecules with subsequent in situ chain stripping from partially acetylated PVA [19]. Also, the same phenomenon can be expected from pectin substances present in SDF (iii) 312–425 °C, chain scission and pyrolysis of PVA and SDF. Comparing the relevant values for both individual PVA (302 °C) and rice bran (230 °C) the thermal decomposition value of NF has been shifted to 312 °C indicating an extra thermal stability in the composite.
Thermal properties of synthesized CSNFP
CSNFP exhibited endotherms at 150 and 222 °C which are due to the melting of SDF and PVA components in the composite and at 340 °C which is due to the subsequent decomposition (Fig. 4).
As can be seen in Fig. 4, the endotherm corresponding to the melting of the SDF at 110 °C in NF has been shifted to a higher temperature (150 °C) in CSNFP, indicating a better interaction between pectin, cellulose and other biopolymers and stronger interaction in between NF due to the cross-linking effect. This can be clearly seen in cross-linked NF (CNF) which is working as the control experiment for CSNFP.
Similarly, the melting corresponding to the PVA component has been shifted into a higher value in CSNFP (222 °C) and CNF (220 °C) compared to NF (215 °C). This is due to the same reason explained above. Decomposition temperatures of the fibers also have increased accordingly from 323 to 340 and 346 °C.
The melting point of phytase enzyme also has been shifted from 120 to 150 °C in CSNFP, which can be considered as a significant increase of the physical thermal stability of the enzyme. This could be again due to the strong interactions between the enzyme molecules and functional groups in NF and also due to cross-links in between enzyme molecules in the composite. First mentioned interactions could be assigned as strong hydrogen bonds between surface –OH groups in NF and –NH groups and –C=O groups in the enzyme molecules.
DTA data indicates two regions of maximum weight losses in NF at 312 and 423 °C, while in CSNFP there are three regions at 349, 421 and 438 °C (Fig. 5).
These can be attributed to the corresponding thermal decomposition. The thermal decomposition step at 312 °C in NF has been shifted to 348 °C in CSNFP. This is due to the thermal stability attained by the cross-linking of fibers with STPP. Due to the cross-linking, fiber strands are held together strongly making it resistant to thermal decomposition. This observation is further assisted by the decomposition pattern of CNF. It shows three decomposition steps of CNF at 356, 425 and 432 °C are higher than the values of NF which can be attributed to the proof of concept.
The thermal decomposition step at 238 °C in phytase enzyme cannot be seen in the CSNFP. It has been shifted to 348 °C, which is a significant thermal stabilization of the enzyme. This could be due to the strong hydrogen bonds between surface hydroxyl (OH) groups in NF and amine (NH) groups and carbonyl (C=O) groups in the enzyme molecules. Cross-links between enzyme molecules–enzyme molecules, enzyme molecules–NF, and NF–NF through STPP might have also affected the thermal stability of the enzymes.
Determination of the activity along with thermal stability of CSNFP
The activity of CSNFP was found as 0.1 units/mg. An extra thermal stability of the enzyme was expected due to the multi-point or multi-subunit immobilization to the fiber surface [28].
For phytase enzyme, the optimum working temperature is around 40–45 °C [29]. As the temperature continues to rise above the optimum, the activity and the rate of the reaction decrease abruptly due to the denaturation of enzyme structure. In this study, we observed that for free phytase enzyme the activity was entirely lost around 70–80 °C (Fig. 6).
This observation compares well with the value reported by Wyss et al. [30], as they have observed a complete denaturation of the phytase enzyme extracted from A. niger at temperatures between 50 and 70 °C [30]. Further, it has been reported that the denaturation takes place during the palletization process at temperatures around 70–80 °C [10, 11], which was also evidential in our study.
In contrast to free enzyme, the CSNFP have shown a significant improvement in the thermal stability as it continued to show the enzymatic activity up to 170 °C, an improvement by 100 °C.
Generally, there are two processes responsible for the activity loss of an enzyme with temperature. First, denaturation which usually happens because of the loss of tertiary (and often secondary) protein structure which is not involved in covalent bond cleavage which is theoretically reversible. Second, the degradation which is the loss of primary structure, associated with covalent bond cleavage and/or formation, which is irreversible [31]. Either one or both can account for the inactivation of the enzyme at elevated temperatures, usually above 80 °C. The upper-temperature limit for enzyme stability depends on the conformational integrity of the enzyme or protein. Greater the conformational integrity, greater is the stability. Greater thermal stability of the surface modified enzyme carrying NF may be due to the immobilization of enzymes on the nanofiber surface. Strong hydrogen bonding can be expected between the surface –OH groups present in synthesized NF and free –C=O and –NH groups present in the phytase enzyme. PVA and the dietary fibers are responsible for the surface –OH groups in the NF. Subsequent cross-linking with STTP may have further increased the thermal stability. Strong hydrogen bonding between –O–P groups in STTP and free –NH groups in enzyme and –OH groups in the nanofiber surface a responsible for stable cross-links between enzyme/enzyme, enzyme/NF, and NF/NF.