The Interplay Between Whey Protein Fibrils With Carbon Nanotubes Or Carbon Nano-Onions Part 2
Aug 12, 2024
2.4. Characterization
Scanning electron microscopy (SEM): The surface morphology and structure of the sample were analyzed using a JSM-7100F scanning electron microscope (JEOL, Tokyo, Japan).
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The SEM photos were clearer after being sprayed with gold for 10 min before observation using a transmission electron microscope (TEM, JEM-2010, Tokyo, Japan). The sample was diluted and ultrasonically dispersed. A solution droplet was put onto a carbon support film on a copper grid.
After 15 s, the excess part was removed with filter paper. Subsequently, a droplet of 2% uranyl acetate was put onto the grid and again removed after 15 s. Electron micrographs were taken using a JEOL electron microscope (JEM-2010, Tokyo, Japan) operating at 100 kV.
Fourier transform infrared spectrum (FTIR): A Fourier transform infrared spectrometer (Nicolet iS10, Thermo Fisher Scientific, Waltham, MA, USA) was used. The composite material and potassium bromide were weighed at the mass ratio of 1:100 and ground under an infrared lamp for 10 min to make them evenly mixed.
After compression, the FTIR spectra were recorded. The scanning range was 400~4000 cm−1 and the resolution was 4 cm−1. X-ray diffraction (XRD): The crystal structures of the composites were characterized using a MAXima-X XRD-7000 X-ray diffractometer (Tokyo, Japan) with the following settings: Cu Kα- ray, 40 kV, 2θ from 5◦ to 80◦. Raman spectroscopy: Raman spectra were determined on a HORIBA HR800 (Paris, France) with a 514 nm laser.
Thermogravimetry (TG): The thermal stability of the composites in air was characterized using a NETZSCH STA449 F3 synchronous thermal analyzer (Selb, Germany). The heating range was from 30 to 700 ◦C and the heating rate was 10 ◦C/min.
3. Results and Discussion
3.1. WPI Fibrils
The WPI-1 (without lecithin) fibril solution was transparent and colorless (Figure 1(a1)). The fibrils could be observed through the birefringence of polarized sheets. The WPI-2 (with lecithin) fibril solution was brown (Figure 1(a2)).
Due to their dark color, it was difficult to observe the fibrils via the birefringence sheets. Wang et al. reported that their whey protein concentrate (WPC, containing lecithin) fibril solution gradually changed from transparent light yellow to dark brown within 5 h (80 ◦C, pH 1.8).
They believed that a Maillard reaction occurred, since small peptides formed by WPC hydrolysis during the formation of the fibrils [68]. In this study, the WPI solutions with or without lecithin were both used to prepare the WPI fibril solution.
This is the first time anyone has proven that browning was not due to a Maillard reaction with peptides, while lecithin was the reason for the browning of WPI in the preparation of fibrils.
The TEM results for WPI-1 (protein mass fraction of 97.80%, without lecithin) and WPI-2 (protein mass fraction of 90.39%, containing lecithin) fibrils are shown in Figure 1b,c. It can be observed that fibrils were randomly distributed in the solution.
The length of the WPI fibrils was about 2 µm. Mantovani et al. evaluated the effects of soybean lecithin on the formation of whey protein fibrils. During heat treatment, soybean lecithin had no significant effect on the fibril formation rate or protein secondary structure conformation [69].
The results in Figure 1c show that the fibrils prepared using WPI containing lecithin had a certain agglomeration and dark color, indicating that lecithin may adhere uniformly to WPI fibrils, making the color of the fibril solution darker.
This is consistent with the previous observation that lecithin can darken the color of WPI.
3.2. CNTs and CNOs
Figures 2a and b show the TEM and HR-TEM images of CNTs, respectively. The diameter of CNTs was about 30 nm, with multi-layered graphite walls. The La2NiO4 catalyst was reduced by hydrogen before methane cracking.
After reduction, the "--La--Ni--La-- Ni--" ordered structures were formed on the perovskite-like catalyst surface (: oxygen vacancy). The oxygen vacancy provided a place for methane adsorption on the surface.
The cracking of methane was then found to occur on Ni sites near the oxygen vacancy. The structure of --La--Ni--La--Ni-- inhibited the aggregation of Ni particles and ensured the existence of a high concentration of nanometal Ni catalysts on the surface. Nano-Ni was a necessary condition for the growth of CNTs [70].
Figures 2c and d show the TEM and HR-TEM images of CNOs, respectively. After purification, some carbon onion cores became hollow. The hollow cores measured approximately 100 nm in diameter. The HR-TEM images clearly showed the multi-layer graphitized structure of the CNOs. The Fe-Ni alloy was the nucleation center of the carbon nano-onion formation. Methane was first decomposed into carbon atoms on Fe-Ni.
Carbon atoms penetrated the alloy to form metal carbides. Around the metal carbide catalysts, methane was further cracked and formed a multi-layered graphitic structure [67].
From the HR-TEM images, it was observed that in CNTs, the graphitic layers are not exactly parallel to each other, indicating the existence of defects. In CNOs, some graphitic carbon shell networks were not perfectly closed, indicating the existence of more defects.
3.3. WPI Fibril–CNT (CNOs) Composites
In general, WPI fibril–CNT (or CNO) composites showed relatively uniform colloidal structures, as seen in Figure 3. Because of the highly hydrophobic surfaces of CNTs and CNOs, they were difficult to spontaneously disperse in water in their original forms.
Protein fibrils were amphiphilic, which could effectively adsorb and bind to the graphite surfaces of carbon nanoparticles, providing the required water solubility and biocompatibility [71,72].
Since the whey protein fibrils were also amphiphilic, this helped to solve the dispersion problem related to CNTs and CNOs.
For the of WPI fibril–CNT sample (CNTs: 0.05 wt.%), as seen in Figure 3a, a few agglomerated CNT particles were observed in the colloidal. Some studies reported that whey protein could be an efficient and selective dispersant for CNTs of certain diameters.
The possible active binding sites on the whey protein surfaces had a better match with certain CNTs' curvatures [54]. It was speculated that in the composites with higher concentrations of CNTs, aggregations might occur.
With the addition of more CNTs or CNOs, the viscosity of the composites increased. After drying of the WPI fibril–carbon nanocomposite gels, WPI fibril–CNTs were less uniform but glossier than WPI fibril–CNOs (Figure 3c,f).
The WPI fibril–CNOs could be ideal functional bio-film materials. From Figure 3a,d, it can be seen that WPI fibril–carbon nanomaterials were all evenly gelled. Before the carbon nanomaterials were added, the WPI fibril solutions were not gelatinous at this protein concentration. Neither the individual CNTs nor CNOs were gelatinous in a water solution.
Without a hydrothermal process, the mixtures of WPI fibrils and CNTs (WPI fibrils and CNOs) were not gels. Only when subjected to a hydrothermal process did the composites become colloidal. Some authors have reported that the amyloid fibril-based hydrogels could be altered in terms of both the physical and structural properties in the presence of CNTs [73].
This means that protein fibrils and CNTs interacted under certain conditions. The gel formation might be due to the following factors: (i) the fibrillar structure of WPI fibrils could promote gel formation; (ii) the heating and pressure during the hydrothermal process in the autoclave might help the composite gelatinate; (iii) carbon nanomaterials have negatively charged surfaces, which would interact with the positively charged protein fibrils to form gels, suggesting the possibility of film formation [32]. Figure 4a,e shows the SEM images of WPI fibril–CNTs and WPI fibril–CNOs.
The morphology of the dispersed CNTs and CNOs can be observed. The dispersion of WPI fibril–CNOs (Figure 4e) was better than WPI fibril–CNTs (Figure 4a), supporting the information in Figure 3. In the TEM images of WPI fibril–CNTs (Figure 4b) and WPI fibril– CNOs (Figure 4f), WPI fibrils and CNTs can be observed; similarly, WPI fibrils and CNOs also existed.
No obvious damage was observed in CNTs or CNOs after hybridization with WPI fibrils (Figure 4c,g). However, a significant reduction in the length of WPI fibrils in the composites can be seen in Figure 4d,h.
The lengths of WPI fibrils were shortened from 2 µm to about 200 nm in both the WPI fibril–CNT and WPI fibril–CNO composites. The short fibrils formed small clusters.
The possible reasons for this are as follows: (i) the destruction of the intermolecular force of the fibrils under steam pressure in the autoclave; (ii) the Brownian motion of carbon nanoparticles under pressure also might cause the WPI fibrils to break down; (iii) the β-folded fibril bundles near the turning point of WPI fibrils were distorted and destroyed [74,75].
These results indicate that CNTs and CNOs might destroy WPI fibrils and inhibit further protein fibrosis under hydrothermal conditions. This finding might have important research value in the future in targeted therapy of organ fibrosis and in vivo protein fibrosis.
By using molecule simulation, researchers reported that carbon nanotubes and fullerene prevented the secondary structure formation of amyloid-β peptide oligomers [76–78]. Figure 5 shows the FTIR results for the WPI fibril–carbon nanocomposites.
In general, it was clear that the functional group signals on the WPI fibril–CNOs were stronger than those on the WPI fibril–CNTs, demonstrating a stronger interaction between WPI fibrils and CNOs.
This might be beneficial to the dispersion of CNOs and to forming a homogeneous gel. This result was consistent with the visual observation. The stretching vibration peak of the hydroxyl group appeared at 3500 cm−1, and the stretching vibration peak of N–H of the amide I band appeared at about 3280 cm−1. The peak between 3000 and 2800 cm−1 was the stretching vibration of the C–H bond.
The absorption band in 1400–1300 cm−1 could be attributed to the variable angle vibration of the C–H and C–OH vibrations. The range of 1260~1000 cm−1 was caused by C–OH stretching vibration. In an acidic aqueous solution, it was easier for CNTs and CNOs to carry hydroxyl groups on the surface [79].
The characteristic peaks of FTIR spectra could be used to analyze not only the functional groups of the composites but also the secondary structures of the proteins.
It can be seen from Figure 5 that the vibration types of the amide band were as follows: stretching vibration peak of amide I band C = O (1640 cm−1 ), bending vibration of amide II band in N-H plane, and characteristic absorption peak of C–N stretching vibration (1570–1520 cm−1 ).
The peak patterns of amide I bands and II bands were not affected by the side chain structure of the protein, but rather only by its secondary structure. The change in the protein secondary structure was analyzed by comparing the spectra of the amide I band region [80]. The amide II band sensitively reflected intermolecular or intramolecular hydrogen bond association.
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