Sunflower stem pith cellulose with different allomorphic nanocrystals for oil-in-water emulsions

doi:10.21203/rs.3.rs-5304466/v1

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Sunflower stem pith cellulose with different allomorphic nanocrystals for oil-in-water emulsions

https://doi.org/10.21203/rs.3.rs-5304466/v1

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Nanocellulose (CNF) as a stabilizer for Pickering emulsions has been widely interested in food, cosmetics and pharmaceuticals. However, cellulose crystal types have been less studied on the emulsification properties of Pickering emulsions. In this study, three different crystalline nanocelluloses (CNF I, CNF II and CNF III) were investigated as stabilizers for Pickering emulsions. CNF I was prepared by TEMPO-mediated oxidation. CNF II and CNF III were obtained by immersing CNF I in sodium hydroxide and ethylenediamine, respectively. CNF I was a fibrous structure (L/D=95.29), while CNF II and CNF III appeared as ellipsoidal nanoparticles with a “Needle-like” structure. The aspect ratios-averaged were 21.86 and 44.05, respectively. Three types of CNF had lower zeta potentials (<-30.0 mV). However, Pickering emulsions stabilized by CNF II had smaller droplet sizes (D3, 2), approximately one times smaller droplet sizes of CNF I and CNF III. However, compared with CNF I and CNF III, Pickering emulsions prepared with CNF II had poor stability. It was shown that the crystal morphology of CNF was particularly important for the stability of Pickering emulsions compared to the morphologies of CNF themselves in this work. It is obvious that CNF I and CNF III tended to have better emulsification properties.

Nanocellulose

Allomorphic nanocrystals

Emulsion stability

Pickering emulsions

Pickering emulsions have the advantages of less stabilizer and better emulsion stability than conventional surfactant-stabilized emulsions, they also avoid the use of surfactants and reduce pollution to the environment (Albert, Beladjine, Tsapis, Fattal, Agnely & Huang, 2019; Liu et al., 2018). As a result, it has attracted the interest of research scholars in recent years. Colloidal particles for Pickering emulsion studies have focused on the use of inorganic particles, including silica, calcium carbonate, and montmorillonite (Jafari, Sedaghat Doost, Nikbakht Nasrabadi, Boostani & Van der Meeren, 2020). Compared to inorganic particles, organic particles (especially soft particles such as microgels, proteins and bacteria) are more flexible for Pickering emulsion preparation due to their interfacial activity, deformability, oil-water compatibility, customized functionality, and so on (Brugger, Vermant & Richtering, 2010; Destribats et al., 2011). With the continuous development of research, particles of biomass origin, due to their non-toxicity, renewability and good biocompatibility, have led to a wide range of applications of emulsions prepared from them in industries such as food, drugs, and pharmaceuticals (Ji & Wang, 2023; Marques et al., 2024).

Sunflower is the third largest source of vegetable oil. Large-scale cultivation produces large amounts of lignocellulosic residues. Sunflower stem pith (SSP), a major by-product of sunflower oil production, is a rich source of cellulose (about 40%) (Yan, Wu, Zhou, Luo & Jiang, 2024; Zhang, Cheng, Ma, Zhou & Xu, 2021). However, how to utilize SSP cellulose at high value has been a problem for researchers. The use of SSP cellulose nanoparticles as particle stabilizers in Pickering emulsions has great potential for development and also solves the problem of low utilization of SSP (Lu, Li, Ge, Xie & Wu, 2021; Niu et al., 2023).

Currently, cellulose-based particles are used to stabilize different types of emulsions, including O/W, W/O and complex emulsions. The main types of cellulose-based particles used are CNCs, CNFs and BCs as well as hydrophobically modified nanocellulose (Chakrabarty & Teramoto, 2021; Ji & Wang, 2023). It has been shown that the source, morphology, and apparent charge density of cellulose particles affect the properties of emulsions. Nanocellulose with different crystalline forms has different morphology, crystalline form, surface chemistry, etc. Therefore, when used as particle stabilizers in Pickering emulsions, they may exhibit differences in emulsification properties, which in turn may have an effect on the performance of the emulsion (Dai et al., 2020; Yuan, Zeng, Wang, Cheng & Chen, 2021; Zhang, Wang, Liu & Tang, 2022). Therefore, an in-depth study of the emulsification properties of nanocellulose with different characteristics will be a very meaningful work to explore the mechanism of nanocellulose to stabilize Pickering emulsions and thus enhance the stability of emulsions.

Three types of CNF with different crystalline forms, labeled CNF-I, CNF-II and CNF-Ⅲ, were first prepared. Three different crystalline forms of nanofibrillated cellulose were then used to stabilize the O/W type of Pickering emulsion. To investigate the emulsification performance, emulsion properties and stabilization mechanism of Pickering emulsions stabilized by cellulose nanoparticles of different crystalline types as particle stabilizers. This study can further deepen the understanding of the mechanism of CNF in stabilizing Pickering emulsions, and also guide the selection of appropriate crystalline nanocellulose as particle stabilizers for stabilizing Pickering emulsions. This study can further deepen the understanding of the mechanism of CNF stabilizing Pickering emulsions, and can also guide the selection of suitable crystalline nanocellulose as particle stabilizers.

2.1. Materials

The SSP was acquired from the Baoding, Hebei, China. Cellulose production process included sulfuric acid (H₂SO₄) and sodium chlorite (NaClO₂) hydrolysis to remove hemicellulose and lignin, as described previously (Yan, Wu, Zhou, Luo & Jiang, 2024). Sodium hypochlorite (NaClO), NaClO₂, sodium hydroxide (NaOH), ethylenediamine (EDA), H₂SO₄ and sunflower oil were purchased from Nanjing Chemical Reagent Co. Ltd., Nanjing. 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO), sodium bromide (NaBr) were obtained from Sigma.

2.2 Preparation of different cellulose crystallites

The production process of SSP nanocellulose was carried out by TEMPO/NaBr/NaClO oxidation with NaClO of 3.8 mmol/g-pulp in water at pH 10, as described previously (Okahashi et al., 2021). The oxidized solid was placed in a dialysis bag (MW = 14 kDa) to obtain a fibrous cellulosic solid residue. The resulting residue was mechanically treated in a high-pressure homogenizer (UH-20, Union-Biotech, China). CNF was obtained by passing through the homogenizer 3 times at a pressure of 500 bar.

CNF II and CNF III were obtained by treating CNF I with NaOH and EDA, respectively. 1 g of CNF I was placed in 50 mL of 18.5 wt% NaOH solution at room temperature for 1.5 h. The excess NaOH was then removed by dialysis in distilled water, and the resulting solids were freeze-dried to obtain CNF II. 1g CNF I was immersed in 50 mL of ethylenediamine and reacted at room temperature for 24 h. The reaction was then dialyzed with methanol until the ethylenediamine was completely removed, and the excess methanol was drained under reduced pressure with a vacuum pump, and the product was freeze-dried to obtain CNF III.

2.3 Characterization of different cellulose crystallites

The morphology of CNF samples was observed using transmission electron microscopy (TEM, JEM-1400, Japan). The diluted suspension of CNF samples (0.01%) was deposited on a copper mesh for 10 h to remove water. A UV-1800 spectrometer (Shimadzu, Japan) was used to detect the transmittance of the CNF samples suspension (0.05%) in the wavelength range of 200–900 nm. The surface charge and particle size of the samples were measured by a Zetasizer Nano S90 masterbatch (Malvern Instruments, Britain). The X-ray diffractograms of the CNF samples were examined by an Ultima IV diffractometer (XRD, Tokyo, Japan) with a 2θ range of 5–35°.

2.4 Preparation of Pickering emulsions

Pickering emulsion was prepared by ultrasonic emulsification (KSB-500, China) with CNF suspension (CNF I, CNF II and CNF III) as particle stabilizer and sunflower oil as oil phase. Sunflower oil (10%, 30%, 50% and 70 v/v%) was added to aqueous solutions containing different concentrations of CNCs (0.1, 0.15, 0.2 and 0.3 wt %) utilizing an ultrasonic breaker to sonicate for 2 min to obtain Pickering emulsion samples.

2.5 Characterization of Pickering emulsions

The stability of the emulsion was determined by the emulsion index (EI) and the droplet size, the EI was calculated by Eq. (2) and the surface-weighted mean diameter of the droplet size (D_3,2) was determined by Eq. (3):

$$\:EI=\frac{{H}_{y}}{{H}_{t}}\times\:100$$

$$\:{D}_{\text{3,2}}=\frac{{\sum\:}_{i}{{n}_{i}{d}_{i}}^{3}}{{\sum\:}_{i}{{n}_{i}{d}_{i}}^{2}}$$

where H_y and H_t represented the height of the cream layer and the total height of the emulsion, respectively. n_i was the number of droplets with diameter d_i.

The rheological properties of emulsions were measured by an RS6000 rheometer (HAAKE, German) with a cone (TMP35) and plate (C35/1° Tit) measuring system at 25°C (Luo, Huang, Zhou & Xu, 2021). The morphological changes of droplets over time were observed by optical microscope (Carl Zeiss, Germany).

3.1 Structure of CNF suspensions

CNF I was produced by TEMPO oxidation of SSP cellulose. As shown in Fig. 2(a), the XRD diffraction pattern of CNF I had a strong crystalline diffraction peak at 2θ = 22.1°, which corresponded to the (200) crystallographic plane of the cellulose I. Two relatively weak diffraction peaks at 2θ = 14.7° and 16.4°, represented the (1–10) and (110) reflection planes of the cellulose I structure, respectively (French, 2014; Li, Anankanbil, Pedersen, Nadzieja & Guo, 2023). CNF I was treated with NaOH (18.5 wt%) to obtain CNF II samples with obvious crystallographic diffraction peaks at 2θ = 12.3°, 20.1°, and 21.8°, which corresponded to (1–10), (110), and (020) reflection planes in the crystalline structure of typical cellulose II, respectively (Yue et al., 2012). This indicated the successful preparation of CNF with cellulose type II. Cellulose type III was obtained by impregnating CNF I with anhydrous EDA at room temperature. From the XRD spectra of CNF III, it could be found that the diffraction peaks at 2θ = 12.1°, 16.6° and 21.5° corresponded to the reflection planes (010), (002) and (100) of typical cellulose III structure, respectively (French, 2014).

Carboxylation increased the surface charge, which was essential in the application of nanocellulose materials. Several studies reported that nanoscale materials should possess higher zeta potentials and increase their degree of dispersion to prevent aggregation in suspensions. As expected, the zeta potential of CNF I was − 55.3 mV, much higher than that of CNF II (-30.0 mV) and CNF III (-37.6 mV) in Table 1. Visual imaging of the suspensions was performed after 4 h of resting. The stability of CNF I was greater than that of CNF II and CNF III was completely placed after 4 h (Fig. 2b). The transmittance of three suspensions of CNF was shown in Fig. 2b. Compared with CNF II and CNF III, CNF I with a higher surface charge had a higher transmittance of 80.1% at 800 nm and was well dispersed in aqueous solutions. Thus, the dispersion stability and transmittance depend greatly on the surface charge of CNF (Luo, Huang, Xu & Fan, 2019).

To further study the morphology of CNF I, CNF II and CNF III, TEM images and corresponding length distribution were compared in Fig. 2c and Table 1. CNF I, CNF II and CNF III particles exhibited good dispersion, but there were significant differences in particle morphology and size. The particles of CNF I showed a fibrous structure with aspect ratios of approximately 95.29. The results for morphology and size were consistent with CNF reported in other studies (Wang et al., 2025). In contrast, the particles of CNF II and CNF III appeared as a “Needle-like” structure. The aspect ratios-averaged were 21.86 and 44.05, respectively, for CNF II and CNF III. Since the particle size analysis by the particle size analyzer was based on a spherical model, the results obtained for the measurement of non-spherical particles did not really reflect the size of the particles numerically. However, particles of the same type could reflect the relative size relationship of the particle size. The results showed that the particle size of CNCs-I, CNF II and CNF III was 2016.2 nm, 620.8 nm and 1162.1 nm. Due to the small zeta potential of CNF II and CNF III, slight flocculation occurred by a Zetasizer Nano S90 masterbatch. The measured particle size dimensions are actually the particle size of the nanocellulose particle flocs. However, the particle size of CNCs-I was still slightly larger than that of CNF II and CNF III. The result was consistent with the TEM analysis.

Table 1

List of morphological and physicochemical characteristics of CNF I, CNF II and CNF III samples.
Sample	Particle diameter (nm)	CNF charge (mV)	Transmittance at 800 nm (%)	Length (nm)	Width (nm)	Aspect ratios
CNF I	2016.2	-55.3	80.1	1125.32	11.81	95.29
CNF II	620.8	-30.0	64.5	189.53	8.67	21.86
CNF III	1162.1	-37.6	76.1	470.47	10.68	44.05

3.2. Characterization of different CNF stabilized emulsions

The emulsification properties of CNF I, CNF II and CNF III particles exhibited significant differences. The effects of different loading (0.1, 0.2, and 0.3 wt%) of CNF I, CNF II and CNF III on the stability of emulsions (oil/water volume ratio 1/9) were studied. As shown in Fig. 3(a, c), the EI of emulsions increased with increasing concentration for emulsions prepared by CNF I, CNF II and CNF III, respectively. When the concentration of CNF was 0.1 wt%, the EI of emulsions prepared from CNF I, CNF II and CNF III were approximately 33.6%, 30.7% and 33.1%, respectively. As the concentration was greater than 0.1 wt%, the EI of the emulsions increased rapidly with the increase in concentration. The EI of the stabilized emulsions of CNF I, CNF II and CNF III were about 95.3%, 84.3% and 98.8%, respectively, when the concentration reached 0.3 wt%. The phenomenon indicated that higher cellulose concentration, which was conducive to improved emulsification properties. Moreover, we also found that the EI of CNF I or CNF III -stabilized emulsions was always higher than that of CNF II -stabilized emulsions at the same CNF content. Due to the lower zeta potential of CNF I (-55.3 mV) and CNF III (-37.6 mV) compared to CNF II (-30.0 mV), it enhanced electrostatic repulsion and formed more stable emulsions(Li, Li, Gong, Kuang, Mo & Song, 2018; Zhou, Sun, Bei, Zahi, Yuan & Liang, 2018). Moreover, when the amount of CNF II was 0.1–0.2%, the oil phase was completely stratified after 14 d of storage (Fig. 3b), indicating that the stability of the emulsion was low. When the CNF II loading was increased to 0.3 wt %, the oil phase did not appear in the upper layer. When CNF I or CNF III was used to stabilize the emulsions, the EI of the emulsions increased with the increase in the amount of CNF I or CNF III after 14 d of storage. When the amount of CNF I and CNF III was 0.2%, the EI of the emulsions were 85% and 62.9%, respectively. Possibly due to the higher aspect ratio and lower charge of CNF I and CNF III, the formation of Pickering emulsions can be promoted (Li et al., 2019; Ma et al., 2023).

To further study the emulsion stability of CNF I, CNF II and CNF III, their Zeta potential and particle size were analyzed. The stability of CNF suspensions at different surface charges was important for their application as emulsion stabilisers (Bao et al., 2021). The zeta potentials of emulsions formed by CNF suspensions at different concentrations were shown in Fig. 4a. The results showed that CNF I and CNF III concentrations had a significant effect on the zeta potential of their stabilized emulsions. When CNF I and CNF III concentrations were increased from 0.1 to 0.3 wt%, the zeta potentials of CNF I-and CNF III-stabilized emulsions decreased by 14.8 and 16.2 mV, respectively. However, the zeta potential of CNF II-stabilized emulsions did not change significantly at different concentrations of CNF II (approximately − 15.3 mV). The result indicated that CNF II with high zeta potential was not favorable for the preparation of stable emulsions. The particle size and morphology of CNF-stabilized emulsions were further analyzed (Fig. 4b). As the amount of CNFs increased, the emulsion particle size decreased. This is also demonstrated by microscopic observation of the emulsion particle size as shown in Fig. 4c. However, CNF-stabilized emulsions showed an increase in the emulsion particle size after 14 d. The CNF I-stabilized emulsions showed a smaller rate of change while the CNF II stabilized emulsions showed a larger rate of change. It was further proved that CNF II was not conducive to the preparation of stable emulsions.

The viscosity of Pickering's emulsion was related to its stability. Viscosity in the continuous aqueous phase increased with CNF I or CNF III content, contributing to oil droplet stabilization (Fig. 5) (Pandey et al., 2018). Overshoot was observed in the low shear rate zone, which was mainly due to the orientation of emulsion droplets under stress. As the surface of the emulsion was covered with CNF I or CNF III, the interaction between CNF resulted in this behavior. Meanwhile, the network formed by CNF I or CNF III was gradually disrupted with the increase in shear force, showing typical shear thinning behavior (Paximada, Tsouko, Kopsahelis, Koutinas & Mandala, 2016). However, we found that the viscosity of CNF II-stabilized emulsions did not increase significantly with the increase of CNF II content, indicating that CNF II was not conducive to the preparation of emulsions.

The influence of the oil volume (10–70% oil) on the emulsion index was investigated (Fig. 6). As shown in Fig. 6a, when the oil phase was increased to 30%, the EI of the emulsion stabilized with CNF I, CNF II or CNF III was 100%, 98% and 86%, respectively. However, the EI of the CNF I and CNF II-stabilized emulsions gradually decreased with the increase of oil volume, and reached 58% and 70% with the 50% oil/water ratio, respectively, which indicated that the CNF I and CNF II tended to form aggregates, and the stability of the emulsion decreased correspondingly in the high oil phase. The emulsion stabilized with CNF III remained better stable under high oil volume (< 70% oil volume). When CNF I, CNF II or CNF III-stabilised emulsions were stored at room temperature for 14 d days (Fig. 6b-c), the EI of the emulsions decreased, indicating that the emulsions were not favorably stabilized. The emulsions stabilized with CNF III were still stable under the oil phase of 30%, inhibiting the emulsion droplet from freely moving and contributing to the improvement of the emulsion stability (Angkuratipakorn, Sriprai, Tantrawong, Chaiyasit & Singkhonrat, 2017; Li et al., 2019). It is noted that the volume of the oil phase was the main factor influencing the stability of the emulsion. As shown in Fig. 7a-c. The emulsion droplet size (D_3,2) of CNF I, CNF II and CNF III-stabilized emulsions increased slightly as the oil phase increased to 70%. The larger droplet size indicated that the emulsion droplets had coalesced. Emulsion was susceptible to flocculation in emulsion systems with high oil phases (Fig. 7d). In addition, with increased storage time, CNF I, CNF II and CNF III-stabilized emulsion droplets coalesced, which was detrimental to the stability of the emulsion. Overall, the high oil phase system may lead to the instability of the emulsion by different allomorphic nanocrystals.

Cellulose was a polysaccharide, and charge density and aggregation were influenced by the crystal structure of cellulose. Therefore, the stability of CNFs in different crystalline forms was crucial for their application as stabilizers in Pickering emulsions. The zeta potentials of CNFs were determined at different crystalline forms, and the results were shown in Fig. 8a. The zeta potential of CNFs was significantly affected by the crystalline form. The zeta potential of emulsions prepared by CNF I and CNF III was higher than that of CNF II, which also suggested that CNF I and CNF III-stabilized emulsions had better stability. When the zeta potential of CNFs changed significantly as the oil volume increased, indicating that the increase in the oil phase influenced the stability of the emulsion. It is noteworthy that the zeta potential of CNF II remained low under different oil phases (approximately − 15 mV), which was unfavorable for the dispersion of CNF II and the stability of food emulsions.

The rheological behavior of the dispersed system under shear stress can effectively reflect its viscoelasticity and provide effective information about the interaction between the dispersed phases. The energy storage modulus G' of the emulsion was consistently greater than the loss modulus G'' (Fig. 8b-d). This was typical gel-like behavior, indicating the formation of an emulsion gel. Meanwhile, the energy storage modulus G' of the CNF I and CNF III-stabilized emulsions increased with the oil phase, indicating the formation of more gel network structures in the emulsion system. In summary, the high oil phase resulted in emulsions that were prone to gel formation. This is mainly attributed to the network structure formed by flocculation between CNF in the high oil phase. However, the G' and G'' of the CNF II-stabilized emulsions were unstable as the oil phase was elevated, indicating that a stable network structure was not formed.

We explored the stabilization mechanism of CNF as a stabilizer for Pickering emulsions. CNF were prepared from sunflower stem pith cellulose. Three different crystalline nanocelluloses (CNF I, CNF II and CNF III) were investigated as stabilizers for Pickering emulsions. CNF I was prepared by TEMPO-mediated oxidation. CNF II and CNF III were obtained by immersing CNF I in sodium hydroxide and ethylenediamine, respectively. The three types of cellulose were investigated for their different influences on the properties of Pickering emulsions. CNF I was a fibrous structure (about 1125.32 nm in length), while CNF II and CNF III were needle-like particles (189.53 nm in length and 470.47 nm in length). The result showed the emulsion ratio of Pickering emulsions stabilized by CNF I and CNF III were higher than that by CNF II. CNF I and CNF III showed a higher aspect ratio, which produced a highly stable emulsion. Pickering emulsions stabilized by CNF II presented about 1-time smaller droplet sizes than CNF I and CNF III. However, with prolonged placement time, CNF II-stabilized emulsion droplets ruptured severely were not conducive to storage. It would provide guidance for selecting suitable allomorphic nanocrystals of CNF as stabilizers for Pickering emulsions.

CRediT authorship contribution statement

Tianqi Feng: Investigation, Formal analysis, Methodology, Writing – review & editing. Zhiyun Sun: Methodology, Investigation. Chen Yan: Conceptualization, Methodology, Software. Caoxing Huang: Writing – review & editing. Xin Zhou: Conceptualization, Resources, Supervision, Writing – review & editing. All authors read and approve the final manuscript.

Acknowledgements

This work was supported by the Natural Science Foundation of Jiangsu Province (Youth Program) (BK20210610), the Postgraduate Research & Practice Innovation Program of Jiangsu Province (SJCX23_0339), the Nanjing Forestry University student innovation training program project (202410298015Z), and Qing Lan Project of Jiangsu Province, PR China.

Funding

The authors have not disclosed any funding.

Conflict of interest

The authors declare no competing interests.

Ethical Approval

The authors hereby declare that the manuscript has not been concurrently submitted to any other journal, and that the submitted work is original and has not been previously published in any form. Furthermore, this study is distinct from any other ongoing research, and all results are presented with utmost integrity, without any fabrication, falsification or inappropriate manipulation of data. Additionally, no data, text or theories by others have been presented as our own. This article does not contain any studies with human participants or animals performed by any of the authors.

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No competing interests reported.

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Editorial decision: Revision requested
11 Nov, 2024
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06 Nov, 2024
Submission checks completed at journal
24 Oct, 2024
First submitted to journal
21 Oct, 2024

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Sunflower stem pith cellulose with different allomorphic nanocrystals for oil-in-water emulsions

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Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Materials

2.2 Preparation of different cellulose crystallites

2.3 Characterization of different cellulose crystallites

2.4 Preparation of Pickering emulsions

2.5 Characterization of Pickering emulsions

3. Results and discussion

3.1 Structure of CNF suspensions

3.2. Characterization of different CNF stabilized emulsions

4. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1