Rheology and Stability of Tunicate Cellulose Nanocrystal-Based Pickering Emulsions: Role of pH, Concentration, and Emulsification Method

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors of the manuscript show that tunicate cellulose nanocrystals (T-CNCs), with their rod-like morphology, very high aspect ratio (≈122), and high crystallinity, exhibit distinct physicochemical properties that allow them to stabilize Pickering emulsions at very low concentrations (0.2–0.4 wt%). The stability and rheology of these emulsions strongly depend on pH (via control of surface charge and contact angle) and on the processing method. The T-CNC emulsion at pH 3 obtained through high-shear homogenization combined with ultrasonication shows the smallest droplets, the highest detachment energy, the best 7-day stability, and the strongest rheological profile.

My observations are as follows:

• The authors present T-CNCs as a sustainable alternative to synthetic surfactants; however, the results are limited to 7 days, no investigations were carried out in the presence of food product components, no example of a food product with pH 3 where they could be used was provided, and lastly, food safety concerns may arise due to the method of obtaining T-CNCs.
• Line 100: Styela clava must be written in italics.
• Are both Equations 3 and 4 used for the results? And is Equation 4 written correctly? In the cited article, the formula is mathematically significantly different.
• For TEM, selection criteria and corrections should be specified in order to ensure accurate, representative results and valid interpretations.
• For droplet size measurements, dilution was performed in pH-adjusted water with 30 s ultrasonication. In this case, is there a possibility that the aggregation of hydrophobic T-CNCs may be altered?
• How does 7-day storage influence coarsening? Is the increase due to Ostwald ripening or coalescence?
• In Section 3.2.2 it is stated that ultrasonication slightly reduces the negative charge, contrary to the observations of Meirelles et al. I believe an explanation should be provided.
• Wouldn’t it have been interesting to monitor viscosity also at shear rates higher than 1000 s⁻¹?

Author Response

The authors of the manuscript show that tunicate cellulose nanocrystals (T-CNCs), with their rod-like morphology, very high aspect ratio (≈122), and high crystallinity, exhibit distinct physicochemical properties that allow them to stabilize Pickering emulsions at very low concentrations (0.2-0.4 wt%). The stability and rheology of these emulsions strongly depend on pH (via control of surface charge and contact angle) and on the processing method. The T-CNC emulsion at pH 3 obtained through high-shear homogenization combined with ultrasonication shows the smallest droplets, the highest detachment energy, the best 7-day stability, and the strongest rheological profile.

My observations are as follows:

• The authors present T-CNCs as a sustainable alternative to synthetic surfactants; however, the results are limited to 7 days, no investigations were carried out in the presence of food product components, no example of a food product with pH 3 where they could be used was provided, and lastly, food safety concerns may arise due to the method of obtaining T-CNCs.

Authors’ response: This is the first study of this kind using T-CNC and obviously, more research will be needed to incorporate T-CNC in a food product. In this fundamental research, we wanted to establish the conditions for T-CNC as a Pickering emulsifier in foods. We have already discussed the type of food products with an acidic pH range where these emulsions could be used (Lines 768-769). We have also discussed the food safety concern of T-CNC extraction (Lines 771-774).

• Line 100: Styela clava must be written in italics.

Authors’ response: Fixed. Lines 102, 281 and 750.

• Are both Equations 3 and 4 used for the results? And is Equation 4 written correctly? In the cited article, the formula is mathematically significantly different.

Authors’ response: Only Equation 4 was used in the calculation of the detachment energy presented in the results. We appreciate the reviewer’s observation regarding the mathematical form reported in the cited article. The difference arises because the cited study evaluated the reduction in free energy upon particle adsorption, for which the change in free energy is expressed as a negative quantity since adsorption lowers interfacial energy. In contrast, our study uses the equation to calculate the energy required to detach a particle from the interface, which is defined as a positive energy barrier. Therefore, the sign convention differs between the two formulations.

To avoid ambiguity, we have now referenced both sources from which the detachment-energy expressions for rod-like particles were derived. These equations originate from the work cited in Refs. [31] and [32]. Line 188.

• For TEM, selection criteria and corrections should be specified in order to ensure accurate, representative results and valid interpretations.

Authors’ response: We have expanded the Methods section accordingly. We now clearly describe the criteria used for particle selection (well-isolated CNC particle, intact morphology, absence of staining artifacts) and the procedure for excluding overlapping or bundled structures. These additions ensure that the TEM analysis is transparent and reproducible. Please see section 2.3.1.

• For droplet size measurements, dilution was performed in pH-adjusted water with 30 s ultrasonication. In this case, is there a possibility that the aggregation of hydrophobic T-CNCs may be altered?

Authors’ response: We appreciate the reviewer’s careful observation. In our system, T-CNCs obtained by metal-assisted oxidation are predominantly hydrophilic and carry a negative surface charge under the experimental conditions; therefore, classical hydrophobic aggregation is not expected. However, we acknowledge that weak CNC-CNC associations (e.g., hydrogen-bonded clusters or loosely bridged particles) can exist in the continuous phase before dilution, and these may contribute to the small peak observed around 1 µm in the size distributions.

The dilution procedure and the 30s ultrasonication step used for Mastersizer measurements are identical to those commonly adopted in Pickering emulsion characterization and are intended to gently disrupt loose flocs and reduce multiple scattering. It is likely that this brief sonication partially breaks up CNC-CNC clusters, which would slightly modify the intensity of the minor peak around 1 µm. However, this small peak represents a very low volume fractioncompared with the main droplet population and thus has a negligible influence on the volume-weighted mean diameter (d₄,₃) that we report. The dominant peak, corresponding to the oil droplets (typically > 1 µm), remains essentially unchanged under these conditions, and all formulations were processed and measured using the same protocol. Therefore, even if some restructuring of CNC–CNC aggregates occurs during measurement, it does not affect the comparative analysis of droplet size among treatments nor the conclusions regarding T-CNC-stabilized emulsions. To avoid any misunderstanding, we have clarified in the Methods and Results that a minor peak near 1 µm is present and likely originates from CNC clusters or very small droplets, and that our interpretation is based primarily on the main droplet population. See lines 225-226 and 388-389.

• How does 7-day storage influence coarsening? Is the increase due to Ostwald ripening or coalescence?

Authors’ response: After 7 days of storage, several emulsions showed a moderate increase in mean droplet size accompanied by peak broadening, the emergence of multimodal distributions, and visible creaming in some formulations. These characteristics are consistent with coalescence-driven growth, rather than Ostwald ripening. Coalescence typically produces wider or bimodal droplet-size distributions and visible macroscopic destabilization, whereas our system did not exhibit the uniform, gradual increase in droplet size that is characteristic of Ostwald ripening. Also, Ostwald ripening is not known to occur in vegetable oil-in-water emulsions, as these oils are insoluble in water. So, the observed peak broadening, formation of a new distribution peak, and creaming behaviour indicate that coalescence is the most plausible explanation for the coarsening seen during 7-day storage. To clarify, we replaced the word “coarsening” with “coalescence in line 416.

• In Section 3.2.2, it is stated that ultrasonication slightly reduces the negative charge, contrary to the observations of Meirelles et al. I believe an explanation should be provided.

Authors’ response: We thank the reviewer for this comment. A brief explanation has now been added to Section 3.2.2 to clarify why ultrasonication produced a slight reduction in droplet ζ-potential in our system. As noted in the revised text, our measurements were performed on CNC-coated droplets rather than free CNC suspensions, and probe ultrasonication can induce droplet breakup, thereby increasing interfacial area and reducing the TCNC's surface coverage, leading to a reduction in droplet charge. Lines 473-477.

• Wouldn’t it have been interesting to monitor viscosity also at shear rates higher than 1000 s⁻¹?

Authors’ response: We agree that extending the shear-rate range beyond 1000 s⁻¹ could provide additional information on the high-shear behaviour of T-CNC-stabilized emulsions. In the present study, however, our rheological measurements were deliberately restricted to 0.1–1000 s⁻¹ for two reasons. First, this range covers the shear conditions most relevant to typical food usage and oral processing scenarios and was sufficient to characterize the shear-thinning behaviour and to compare formulations. Second, measurements above 1000 s⁻¹ often introduce experimental artifacts for this type of emulsion, including edge effects where flow becomes non-uniform near the geometry boundaries, frictional heating that alters viscosity, and wall slip caused by poor adhesion of the sample to the plates. So together, these effects reduce the reliability of high-shear data.

Reviewer 2 Report

Comments and Suggestions for Authors

Among the specific improvements requested for the text are the following:
1) Some references are more than 15 years old. They should be more up-to-date. Examples: 12) Dickinson, E. Interfacial Structure and Stability of Food Emulsions as Affected by Protein–Polysaccharide Interactions. Soft Matter 2008, 4, 932, doi:10.1039/b718319d. 3. Arditty, S.; Whitby, C.P.; Binks, B.P.; Schmitt, V.; Leal-Calderon, F. Some General Features of Limited Coalescence in Solid-Stabilized Emulsions. Eur. Phys. J. E 2003, 11, 273–281, doi:10.1140/epje/i2003-10018-6. 5. Binks, B.P. Particles as Surfactants—Similarities and Differences. Current opinion in colloid & interface science 2002, 7, 21–41.

2) The characterization of the PE O/W emulsion using T-CNC should also be included within point b) of the specific objectives of the work.

3) The storage conditions of the emulsion for 7 days must be specified.

4) Equations 5, 6, and 7 must be correctly identified in the text, preferably within the Materials and Methods section.

5) In the discussion of section 3.2.1, "Droplet Size T-CNC-Stabilized," the references are incorrectly numbered.

6) The methods used to adjust pH 3 and 5 are not specified.

Author Response

1) Some references are more than 15 years old. They should be more up to date. Examples:

12) Dickinson, E. Interfacial Structure and Stability of Food Emulsions as Affected by Protein–Polysaccharide Interactions. Soft Matter 2008, 4, 932, doi:10.1039/b718319d.

3. Arditty, S.; Whitby, C.P.; Binks, B.P.; Schmitt, V.; Leal-Calderon, F. Some General Features of Limited Coalescence in Solid-Stabilized Emulsions. Eur. Phys. J. E 2003, 11, 273–281, doi:10.1140/epje/i2003-10018-6.
4. Binks, B.P. Particles as Surfactants—Similarities and Differences. Current opinion in colloid & interface science 2002, 7, 21–41.

Authors’ response: Some of the above-cited references are original and primary references in particle-based emulsions. Even if they are old, they should be cited to give proper credit to the original research. Based on the reviewer’s comment, we have deleted the reference (12) by Dickinson.

2) The characterization of the PE O/W emulsion using T-CNC should also be included within point b) of the specific objectives of the work.

Authors’ response: We have already added the PE characterization under the specific objective (d). Line 97-99.

3) The storage conditions of the emulsion for 7 days must be specified.

Authors’ response: It is already mentioned in the method section under emulsion preparation. See line 217.

4) Equations 5, 6, and 7 must be correctly identified in the text, preferably within the Materials and Methods section.

Authors’ response: We thank the reviewer for noting this. Our manuscript includes equations only up to Equation 5. The mention of Equation 8 was a typographical error and has been corrected by renumbering it appropriately as Equation 5. This equation is now clearly identified in the Materials and Methods section, and all other equations are consistently referenced in both the Methods and the Results and Discussion sections.

5) In the discussion of section 3.2.1, "Droplet Size T-CNC-Stabilized," the references are incorrectly numbered.

Authors’ response: Ref numbers have been corrected.

6) The methods used to adjust pH 3 and 5 are not specified.

Authors’ response: We have added the methods used to adjust pH 3 and 5 in the fabrication of T-CNC-based Pickering Emulsion. Lines 204-207.

Reviewer 3 Report

Comments and Suggestions for Authors

The study can be considered careful and innovative, particularly in its exploration of alternative solutions to synthetic surfactant-based components. The use of tunicate (marine invertebrate)-derived cellulose nanocrystals (T-CNC) in Pickering emulsions highlights the potential of novel and natural sources, which adds significant value to the research.Overall, the study is well planned and effectively characterized with analyses that support the evaluation of its aims and rationale. Although some sections contain overly long sentences, the manuscript does not present any major language-related issues.

Considering all these points, I would be able to recommend the acceptance of the manuscript once the following revisions and clarifications are fully addressed. My suggestions and evaluations are presented below.

 

Title: The title is well chosen and appropriately reflects the content, objectives, and findings of the study.

Keywords: The selection of keywords is appropriate, and avoiding terms that directly repeat the title is a sound choice. However, it would be more suitable to refrain from using abbreviations in the keyword list. I would also like to suggest including some of the analytical characteristics evaluated in the study as additional keywords. For instance, terms such as droplet size and zeta potential may further enhance the discoverability and relevance of the manuscript.

 

Intruduction: The introduction section is very well written. Previous studies, the aim of the research, and the rationale for selecting tunicate (marine invertebrate)-derived cellulose are clearly and adequately detailed. I would also like to suggest including some of the analytical characteristics evaluated in the study as additional keywords. For instance, terms such as droplet size and zeta potential may further enhance the discoverability and relevance of the manuscript.

 

The Materials and Methods section is sufficiently detailed to ensure the reproducibility of the analyses. I have pointed out a few minor aspects that appear to have been overlooked.

 

# Please ensure that the species name Styela clava is written in italics throughout the manuscript. Kindly review the entire text and correct all relevant occurrences (Line 100).

 

# Please revise the expressions such as “2 wt% NaOH” and similar notations throughout the manuscript. For consistency and improved readability, it would be more appropriate to use the format “2% (w/w) NaOH” in accordance with scientific writing conventions. Line 113,94,125, 126, 191, 193 etc. Please check and correct all of them.

# Line  154 correct as (0.001%, w/v)

# Line  154 , Correct as 40 mL with upper letter

# Table 1 appears somewhat unclear and low in quality, likely because it was inserted as an image. Enhancing the table quality would improve readability. For instance, units and parameters could be aligned consistently, and the parameter names should follow a uniform format, with each beginning with a capital letter (see “Detachment Energy” as an example).

 

# For the parameters Detachment Energy and Surface charge of T-CNC dispersions and T-CNC-stabilized Pickering emulsions, could you clarify why only pH 3 and pH 5 were selected? What was the rationale for not assessing the changes at more extreme pH values such as pH 2 or pH 10? If the characterization analyses at pH 3 and pH 5 were selected based on specific findings or considerations during the study design phase, please clearly state this rationale in the relevant section of the manuscript.

 

# Similarly, for the rheological behavior analysis, could you explain why only these two pH values were examined during storage? Providing the rationale for selecting these specific pH levels would strengthen the methodological clarity.

 

# The references used in the study are generally  up-to-date and of high quality.

 

Result and discussion: Overall, the discussion section is adequate. The findings are compared with relevant previous studies, and the underlying mechanisms of the results are explained appropriately.

 

# In particular, please review the long sentences in the conclusion section and revise them into shorter and clearer forms to improve readability.

 

# Please ensure the use of a consistent writing style throughout the manuscript, particularly in section titles and within the main text. The use of uppercase and lowercase letters should follow a standardized format to maintain clarity and uniformity. See (Red), 0.3% (blue), and (green) Line 490-491

In Figure 5, please indicate the units for the x-axis to improve clarity and readability.

 

# The sentences in the conclusion section are quite long, which reduces clarity and readability. Please revise them to be shorter and more concise. Additionally, addressing the limitations of the study and providing suggestions for future research would offer valuable perspective and guidance for subsequent work.

 

Good luck with corrections.

 

 

 

Author Response

The study can be considered careful and innovative, particularly in its exploration of alternative solutions to synthetic surfactant-based components. The use of tunicate (marine invertebrate)-derived cellulose nanocrystals (T-CNC) in Pickering emulsions highlights the potential of novel and natural sources, which adds significant value to the research. Overall, the study is well planned and effectively characterized with analyses that support the evaluation of its aims and rationale. Although some sections contain overly long sentences, the manuscript does not present any major language-related issues.

Considering all these points, I would be able to recommend the acceptance of the manuscript once the following revisions and clarifications are fully addressed. My suggestions and evaluations are presented below.

Title: The title is well chosen and appropriately reflects the content, objectives, and findings of the study.

Keywords: The selection of keywords is appropriate and avoiding terms that directly repeat the title is a sound choice. However, it would be more suitable to refrain from using abbreviations in the keyword list. I would also like to suggest including some of the analytical characteristics evaluated in the study as additional keywords. For instance, terms such as droplet size and zeta potential may further enhance the discoverability and relevance of the manuscript.

Authors’ response: CNC is such a common term that its use as a keyword is essential. We have also updated the keywords by adding the recommended new ones.

Introduction: The introduction section is very well written. Previous studies, the aim of the research, and the rationale for selecting tunicate (marine invertebrate)-derived cellulose are clearly and adequately detailed. I would also like to suggest including some of the analytical characteristics evaluated in the study as additional keywords. For instance, terms such as "droplet size and zeta potential may further enhance the discoverability and relevance of the manuscript.

Authors’ response: new keywords added.

The Materials and Methods section is sufficiently detailed to ensure the reproducibility of the analyses. I have pointed out a few minor aspects that appear to have been overlooked.

# Please ensure that the species name Styela clava is written in italics throughout the manuscript. Kindly review the entire text and correct all relevant occurrences (Line 100).

Authors’ response: We have fixed all such occurrences. See lines 102, 281 and 750.

# Please revise the expressions such as “2 wt% NaOH” and similar notations throughout the manuscript. For consistency and improved readability, it would be more appropriate to use the format “2% (w/w) NaOH” in accordance with scientific writing conventions. Line 113,94,125, 126, 191, 193 etc. Please check and correct all of them.

Authors’ response: We corrected all the typing errors throughout the manuscript.

# Line 154 correct as (0.001%, w/v)

Authors’ response: Fixed. See line 163

# Line 154 , Correct as 40 mL with upper letter

Authors’ response: Fixed, see line 163.

# Table 1 appears somewhat unclear and low in quality, likely because it was inserted as an image. Enhancing the table quality would improve readability. For instance, units and parameters could be aligned consistently, and the parameter names should follow a uniform format, with each beginning with a capital letter (see “Detachment Energy” as an example).

Authors’ response: We have provided the actual editable Table and fixed the issues as suggested.

# For the parameters Detachment Energy and Surface charge of T-CNC dispersions and T-CNC-stabilized Pickering emulsions, could you clarify why only pH 3 and pH 5 were selected? What was the rationale for not assessing the changes at more extreme pH values such as pH 2 or pH 10? If the characterization analyses at pH 3 and pH 5 were selected based on specific findings or considerations during the study design phase, please clearly state this rationale in the relevant section of the manuscript.

# Similarly, for the rheological behavior analysis, could you explain why only these two pH values were examined during storage? Providing the rationale for selecting these specific pH levels would strengthen the methodological clarity.

Authors’ response: We thank the reviewer for raising this point. The selection of pH 3 and pH 5 in this study was intentional and based on the pH ranges most relevant to the potential application areas of T-CNC-stabilized Pickering emulsions. Many targeted products, including acidic beverages, salad dressings, yogurt and sauces, and several cosmetic formulations, typically fall within a pH range of 3-5. Preliminary screening also indicated that T-CNC suspensions and emulsions showed the most promising stability within this acidic window. For this reason, the interfacial characterization (detachment energy and contact angle), surface charge measurements of the emulsion, and droplet-stability analysis were focused on pH 3 and pH 5. More extreme pH conditions (such as pH 2 or pH 10) were not included because they are not representative of our intended product categories and would not provide additional insight into the functional behaviour of T-CNCs in realistic formulations. Similarly, rheological measurements during storage were performed only at pH 3 and pH 5 to maintain consistency with the stability studies and to reflect the same application-relevant pH range. We have discussed this rationale in the manuscript. See lines 207-211.

# The references used in the study are generally up-to-date and of high quality.

Result and discussion: Overall, the discussion section is adequate. The findings are compared with relevant previous studies, and the underlying mechanisms of the results are explained appropriately.

# Please ensure the use of a consistent writing style throughout the manuscript, particularly in section titles and within the main text. The use of uppercase and lowercase letters should follow a standardized format to maintain clarity and uniformity. See (Red), 0.3% (blue), and (green) Line 490-491

Author’s response: Fixed. See lines 518-519.

In Figure 5, please indicate the units for the x-axis to improve clarity and readability.

Author’s response: We have indicated that the x-axis means T-CNC Concentration (%) not only for Figure 5, but also for Figures 7 and 3.

# In particular, please review the long sentences in the conclusion section and revise them into shorter and clearer forms to improve readability.

# The sentences in the conclusion section are quite long, which reduces clarity and readability. Please revise them to be shorter and more concise. Additionally, addressing the limitations of the study and providing suggestions for future research would offer valuable perspective and guidance for subsequent work.

Author’s response: We revised the conclusion to a shorter form, addressed the limitations of the work, and provided suggestions for future research.

Good luck with corrections.