Review Reports

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The manuscript reports the development of orange peel pomace-derived soluble dietary fiber/sodium alginate films reinforced with cellulose nanocrystal/zinc oxide hybrids and evaluates their structural, mechanical, thermal, optical, antibacterial, and blueberry-preservation properties. The valorization of agricultural waste for active food packaging is relevant to the scope of Foods, and the authors provide a broad experimental dataset covering material characterization and practical preservation performance. However, several methodological limitations and insufficiently supported mechanistic interpretations currently weaken the conclusions, particularly regarding the contributions of the individual components, thermal transitions, barrier properties, and preservation mechanisms. I provided the following issues that should be properly addressed:

- Reference 38 already describes orange peel-derived soluble dietary fiber/sodium alginate films reinforced with nanocellulose, while other cited studies investigate cellulose nanocrystal/ZnO hybrids for functional applications. The authors should clarify which limitations of these previous approaches remain unresolved and explain what specific advantages are expected from incorporating ONCC@ZnONP into the previously developed OSDF/SA matrix. A more critical comparison with existing literature would help establish the novelty and scientific contribution of the present study.

- The OSDF/SA/ONCC formulation is included as a reference, but its reported ONCC content is not directly comparable to the ONCC@ZnONP concentrations used in the composite films. Furthermore, no formulation containing ZnO without ONCC is investigated. These limitations make it difficult to distinguish the individual contributions of cellulose nanocrystals and ZnO to the mechanical and antibacterial properties. The authors should report the actual composition of the hybrid filler and clarify the relative amounts of cellulose and ZnO incorporated into each film, ideally providing appropriate control formulations to substantiate the proposed synergistic effects.

- The SEM and EDS results presented in Figure 2 confirm the presence of zinc-containing structures within the synthesized hybrid, but they do not directly demonstrate the homogeneous anchoring of ZnO nanoparticles onto individual cellulose nanocrystals. The flower-like morphology observed by SEM also differs substantially from the needle-like morphology of the original ONCC. Additional elemental mapping or higher-resolution characterization would help establish the distribution and organization of the inorganic component. Furthermore, the proposed electrostatic interactions between ONCC and ZnO, as well as the interfacial hydrogen bonding discussed in Section 3.3, should be supported more carefully, considering the limited evidence provided by the morphological and FTIR results.

- The broad endothermic transitions shown in Figure 4D are assigned to melting and crystallization-related processes without sufficiently excluding contributions from water evaporation or other thermal events. This distinction is particularly important for hydrophilic polysaccharide-based films containing glycerol and residual moisture. The reported glass transition temperatures should also be supported by a clearer identification of the corresponding thermal features. The authors should reconsider the proposed relationships between filler loading, heterogeneous nucleation, crystallinity, and polymer-chain mobility unless additional experimental evidence can substantiate these interpretations.

- Section 3.5 reports an increase of approximately 135°C in the temperature corresponding to 50% weight loss for O/CZ8 compared with OSDF/SA/ONCC. However, the incorporation of an inorganic filler also changes the residual mass fraction and can significantly influence the interpretation of temperatures defined at a fixed percentage of total mass loss. The authors should distinguish changes in the actual thermal degradation behavior of the polymeric matrix from those arising from the inorganic content and consider reporting additional thermal stability parameters, such as degradation onset temperatures or derivative thermogravimetric results.

- Improvements in blueberry preservation are repeatedly attributed to enhanced water vapor and oxygen barrier properties, although neither water vapor permeability nor oxygen permeability is experimentally determined. These properties cannot be established solely from SEM morphology, tensile behavior, or fruit weight loss. The authors should provide quantitative barrier measurements or substantially moderate the proposed mechanisms, particularly the statements concerning reduced oxygen transport, suppression of respiratory activity, and improved moisture retention.

- Although OSDF is introduced as the main film-forming component and alginate as a co-film-forming agent, their respective contributions to film formation, structural stability, and mechanical properties are not adequately discussed in the Introduction. Particular attention should be given to the ionic crosslinking of alginate by Ca²⁺ ions, which are introduced during film preparation but whose role in network formation is not explained. The authors should expand the discussion by considering recent literature on the structural and functional properties of orange peel dietary fibers (https://doi.org/10.1002/jsfa.14186) and on cation-mediated alginate network formation (https://doi.org/10.1002/adfm.202416390), thereby providing a stronger scientific rationale for the selected formulation.

- Section 2.4.7 describes both inhibition-zone measurements and a quantitative plate-counting assay, but the antibacterial results in Figure 5 are presented mainly as photographs without numerical viable bacterial counts. The corresponding CFU values or antibacterial reduction percentages should be reported to substantiate the concentration-dependent activity of the films. The authors should also clarify whether the inhibition-zone diameters in Table 1 include the 6 mm film discs and verify the strain identification reported for Staphylococcus aureus in Section 2.1. Furthermore, the proposed contributions of Zn²⁺ release and reactive oxygen species generation should be presented as possible mechanisms rather than experimentally demonstrated processes.

- Section 2.5 does not specify the number of blueberries included in each treatment, the number of independently packaged samples, or precisely how the films were applied during storage. These details are necessary to assess experimental reproducibility and the reliability of the reported preservation effects. The illumination conditions should also be reported, particularly because the discussion attributes part of the improved preservation to the UV-shielding properties of ZnO. An unpackaged control would provide an additional useful reference for evaluating the contribution of the packaging itself.

- According to Table 2, O/CZ10 produces the lowest blueberry decay rate after 16 days (7.89%), compared with 15.38% for O/CZ8, whereas O/CZ8 shows better performance in terms of weight loss and firmness retention. The authors should explicitly distinguish these different outcomes and clarify the criteria used to identify the optimal formulation rather than presenting O/CZ8 as providing the best overall preservation performance without further qualification.

- Section 3.4 refers to Figure 3B when discussing the XRD results presented in Figure 4B, while Section 3.10 incorrectly refers to Figure 5C for blueberry firmness measurements. The caption of Supplementary Figure S1 also repeats the label (D) for multiple formulations. In addition, Reference 47 does not appear appropriate for supporting the assignment of characteristic ZnO diffraction peaks. The authors should carefully verify the correspondence between citations and the associated statements, revise the figure captions, and correct the recurring grammatical errors throughout the manuscript.

Author Response

 

Comments 1: Reference 38 already describes orange peel-derived soluble dietary fiber/sodium alginate films reinforced with nanocellulose, while other cited studies investigate cellulose nanocrystal/ZnO hybrids for functional applications. The authors should clarify which limitations of these previous approaches remain unresolved and explain what specific advantages are expected from incorporating ONCC@ZnONP into the previously developed OSDF/SA matrix. A more critical comparison with existing literature would help establish the novelty and scientific contribution of the present study.

 

Response 1: Thank you for your suggestion. The following modifications were added to the Introduction (Page 2, Paragraph 3).

1. Limitations of the previous OSDF/SA/ONCC system (Reference [35], originally Reference [38]):

This reference reported OSDF/SA/ONCC films based on navel orange peel soluble dietary fiber/sodium alginate as the matrix and ONCC as the reinforcing phase. In this system, ONCC acted mainly as a physical reinforcing phase. It inherently lacked antibacterial and UV-shielding capabilities. Therefore, it was difficult to actively inhibit food surface microorganisms, which resulted in limited preservation effects on highly perishable fruits.

2. Limitations of reported CNC/ZnO hybrid systems:

Existing CNC/ZnO hybrid studies mostly focused on matrices such as PVA, PLA, PHBV, gelatin, and soy protein isolate. ZnO was often introduced through physical blending. This easily caused agglomeration and led to poor filler–matrix interfacial compatibility, as well as trade-offs among mechanical, optical, and barrier properties. More importantly, these studies did not target the OSDF/SA matrix. They also did not clarify the compatibility between ONCC@ZnONP and the OSDF matrix.

3. Specific advantages of this study:

ONCC served as a green template and dispersant. It improved the dispersion of ZnO and promoted its interfacial compatibility with OSDF. Additionally, ONCC@ZnONP endowed the composite films with UV-shielding and antibacterial properties.

This critical comparison was supplemented in the Introduction.

[35] Chen, L.L.; Wu, Y.C.; Guo, Y.T.; Yan, X.F.; Liu, W.L.; Huang, S. Preparation and Characterization of Soluble Dietary Fiber Edible Packaging Films Reinforced by Nanocellulose from Navel Orange Peel Pomace, Polymers. 2024, 16, 315.

 

Comments 2: The OSDF/SA/ONCC formulation is included as a reference, but its reported ONCC content is not directly comparable to the ONCC@ZnONP concentrations used in the composite films. Furthermore, no formulation containing ZnO without ONCC is investigated. These limitations make it difficult to distinguish the individual contributions of cellulose nanocrystals and ZnO to the mechanical and antibacterial properties. The authors should report the actual composition of the hybrid filler and clarify the relative amounts of cellulose and ZnO incorporated into each film, ideally providing appropriate control formulations to substantiate the proposed synergistic effects.

Response 2: Thank you for your suggestion. The following modifications were made.

1. Hybrid filler composition (added to Sections 2.2 and 2.3):

Based on the synthesis stoichiometry (5 mmol ZnCl₂ → 5 mmol ZnO = 0.4069 g; 29 mL 0.14 wt% ONCC suspension = 0.0406 g ONCC), the theoretical mass ratio of ZnO to ONCC in ONCC@ZnONP was approximately 10:1 (i.e., 90.9 wt% ZnO and 9.1 wt% ONCC). Correspondingly, the O/CZ2–O/CZ10 films contained 1.82–9.09 wt% ZnO and 0.18–0.91 wt% ONCC, respectively.

2. Role of the reference formulation (clarified in Section 2.3):

The OSDF/SA/ONCC film (1 wt% ONCC suspension) served as a qualitative baseline. This demonstrated that ONCC alone did not produce antibacterial activity.

3. Attribution based on existing data:

Neither baseline film (OSDF/SA and OSDF/SA/ONCC) exhibited inhibition zones. Therefore, the antibacterial activity could be clearly attributed to ZnO. The O/CZ films exhibited better mechanical properties than both baselines. Therefore, the mechanical enhancement could be attributed to the hybrid structure.

4. Regarding the ZnO control without ONCC (added to Sections 3.7 and 3.9):

The in situ grown ONCC@ZnONP hybrid was introduced into the matrix as an integrated organic-inorganic dual-functional unit. This endowed it with tight interfacial bonding and high dispersion. Directly comparing it with single ZnO or a simple physical mixture made it difficult to truly reflect the structural advantages of this in situ hybrid. Future studies would supplement single-component or physical mixture control groups on this basis. This would refine the quantitative analysis of individual component contributions.

 

Comments 3: The SEM and EDS results presented in Figure 2 confirm the presence of zinc-containing structures within the synthesized hybrid, but they do not directly demonstrate the homogeneous anchoring of ZnO nanoparticles onto individual cellulose nanocrystals. The flower-like morphology observed by SEM also differs substantially from the needle-like morphology of the original ONCC. Additional elemental mapping or higher-resolution characterization would help establish the distribution and organization of the inorganic component. Furthermore, the proposed electrostatic interactions between ONCC and ZnO, as well as the interfacial hydrogen bonding discussed in Section 3.3, should be supported more carefully, considering the limited evidence provided by the morphological and FTIR results.

 

Response 3: Thank you for your suggestion. The manuscript was revised from three aspects.

1. Morphological reinterpretation (Section 3.1):

The flower-like hierarchical morphology observed by SEM was a micro-nano structure. It was formed by the in situ growth and self-assembly of ZnO on the ONCC template. The difference from the original needle-like ONCC accurately reflected the occurrence of this hybridization process.

2. Characterization limitations:

SEM and EDS results could not analyze the elemental distribution at the single-fiber scale. However, the combination of EDS quantitative composition and FTIR characteristic peak changes was sufficient to support the conclusion of successful hybridization.

3. Weakened mechanistic statements (relevant descriptions in Sections 3.1 and 3.3 were modified).

 

Comments 4: The broad endothermic transitions shown in Figure 4D are assigned to melting and crystallization-related processes without sufficiently excluding contributions from water evaporation or other thermal events. This distinction is particularly important for hydrophilic polysaccharide-based films containing glycerol and residual moisture. The reported glass transition temperatures should also be supported by a clearer identification of the corresponding thermal features. The authors should reconsider the proposed relationships between filler loading, heterogeneous nucleation, crystallinity, and polymer-chain mobility unless additional experimental evidence can substantiate these interpretations.

 

Response 4: Thank you for your suggestion. The following modifications were made.

1. The peak originally assigned to "melting" was redefined as the "apparent melting temperature" (T_m). This acknowledged the overlapping contributions of residual moisture evaporation, glycerol-related thermal events, and polymer melting.
2. T_g was determined according to the equal-area method in ISO 11357-2. It was defined as the apparent glass transition temperature (T_g). The corresponding thermal feature was a baseline step. This was stated in Section 2.4.4.
3. All statements regarding "heterogeneous nucleation" and "crystallinity enhancement" were deleted from Sections 3.4 and 3.7. The shifts in T_g and T_m were discussed based on the interfacial non-covalent interactions between ONCC@ZnONP and OSDF segments, as well as their restriction on segmental motion. This explanation was consistent with the SEM morphology and mechanical property changes.
4. The XRD patterns (Fig. 4B) confirmed that all films maintained an amorphous state. This supported the revised explanation.

 

Comments 5: Section 3.5 reports an increase of approximately 135°C in the temperature corresponding to 50% weight loss for O/CZ8 compared with OSDF/SA/ONCC. However, the incorporation of an inorganic filler also changes the residual mass fraction and can significantly influence the interpretation of temperatures defined at a fixed percentage of total mass loss. The authors should distinguish changes in the actual thermal degradation behavior of the polymeric matrix from those arising from the inorganic content and consider reporting additional thermal stability parameters, such as degradation onset temperatures or derivative thermogravimetric results.

 

Response 5: Thank you for your suggestion. The following modifications were made to Section 3.5 in the manuscript.

1. The TG curves were normalized based on the polymer matrix. This was done to eliminate the interference from the mass of the inorganic filler.
2. The onset decomposition temperature (T_onset, 5% weight loss) and the maximum weight loss rate temperature in DTG (T_max) were added as supplementary evaluation parameters.
3. After normalization, the main degradation stages of all films were located at 200–300°C. The DTG peak temperatures were also similar. This confirmed that ONCC@ZnONP did not significantly alter the intrinsic thermal degradation pathway of the OSDF/SA matrix.
4. The increase in T_50% for the O/CZ8 film was mainly attributed to the higher inorganic residual mass of ONCC@ZnONP. The interfacial non-covalent interactions also contributed to the increased char yield. Statements regarding the effect of "heterogeneous nucleation" on thermal behavior were deleted.

 

Comments 6: Improvements in blueberry preservation are repeatedly attributed to enhanced water vapor and oxygen barrier properties, although neither water vapor permeability nor oxygen permeability is experimentally determined. These properties cannot be established solely from SEM morphology, tensile behavior, or fruit weight loss. The authors should provide quantitative barrier measurements or substantially moderate the proposed mechanisms, particularly the statements concerning reduced oxygen transport, suppression of respiratory activity, and improved moisture retention.

 

Response 6: Thank you for your suggestion. This study indeed did not experimentally measure the water vapor permeability (WVP) and oxygen permeability (OP). Furthermore, barrier properties could not be inferred solely from SEM morphology, tensile behavior, or fruit weight loss data. Therefore, the second option suggested by the reviewer was adopted. This involved substantially moderating the statements regarding the proposed mechanisms. The relevant mechanistic descriptions in Section 3.10 and the Conclusions were comprehensively moderated and revised.

 

Comments 7: Although OSDF is introduced as the main film-forming component and alginate as a co-film-forming agent, their respective contributions to film formation, structural stability, and mechanical properties are not adequately discussed in the Introduction. Particular attention should be given to the ionic crosslinking of alginate by Ca²⁺ ions, which are introduced during film preparation but whose role in network formation is not explained. The authors should expand the discussion by considering recent literature on the structural and functional properties of orange peel dietary fibers (https://doi.org/10.1002/jsfa.14186) and on cation-mediated alginate network formation (https://doi.org/10.1002/adfm.202416390), thereby providing a stronger scientific rationale for the selected formulation.

 

Response 7: Thank you for your suggestion. The Introduction was expanded as follows.

1. OSDF (Wang et al., 2025, newly added citation):

Although OSDF possessed good hydration and film-forming properties (Wang et al., 2025), a single OSDF matrix had low crosslinking density. This restricted its structural stability and mechanical strength.

2. SA (Tordi et al., 2024, newly added citation):

Sodium alginate (SA) was introduced. Its G-blocks coordinated with the added Ca²⁺ during film preparation to form a classic "egg-box" ionic crosslinking network. This enhanced the structural density of the matrix and provided a foundation for filler dispersion and stress transfer.

3. It was clarified that the Ca²⁺-crosslinked SA network served as a fixed base matrix. Therefore, its crosslinking degree was not evaluated as an independent variable.

[1] Wang, Y.R.; Bao, X.D.; Zhao, G.H.; Peng, L.T.; Zhao, F.L.; Qin, Y.B.; Zhang, R.F. Green preparation and evaluation of physi-cochemical, structural and functional properties of dietary fiber from navel orange peel, J Sci Food Agric. 2025, 105: 4515-4523.

[2] Tordi, P.; Ridi, F.; Samorì, P.; Bonini, M. Cation-Alginate Complexes and Their Hydrogels: A Powerful Toolkit for the Development of Next-Generation Sustainable Functional Materials, Adv. Funct. Mater. 2025, 35, 2416390.

 

Comments 8: Section 2.4.7 describes both inhibition-zone measurements and a quantitative plate-counting assay, but the antibacterial results in Figure 5 are presented mainly as photographs without numerical viable bacterial counts. The corresponding CFU values or antibacterial reduction percentages should be reported to substantiate the concentration-dependent activity of the films. The authors should also clarify whether the inhibition-zone diameters in Table 1 include the 6 mm film discs and verify the strain identification reported for Staphylococcus aureus in Section 2.1. Furthermore, the proposed contributions of Zn²⁺ release and reactive oxygen species generation should be presented as possible mechanisms rather than experimentally demonstrated processes.

 

Response 8: Thank you for your suggestion. The following modifications and clarifications were made in response to the above comments.

1. Regarding CFU values:

The plate counting results showed that colony growth in the control groups (OSDF/SA and OSDF/SA/ONCC films) was too dense. It exceeded the reliable counting limit (>300 CFU/plate). Therefore, accurate CFU/mL values could not be provided. The inhibition zone diameter (Table 1) was used as the quantitative evaluation index. Plate photographs (Fig. 5C–P) were used to visually present the colony viability. This combination clearly confirmed the dose-dependent antibacterial trend of the O/CZ films with increasing filler loading.

2. Inhibition zone diameter:

The diameters in Table 1 represented the total diameter, including the 6 mm film disc itself. This was clarified in the main text and in Table 1.

3. Strain identification:

The strain number for Staphylococcus aureus in Section 2.1 was corrected to ATCC 25923.

4. The relevant descriptions in Section 3.9 were revised. These were clearly presented as possible mechanisms rather than experimentally demonstrated processes.

 

Comments 9: Section 2.5 does not specify the number of blueberries included in each treatment, the number of independently packaged samples, or precisely how the films were applied during storage. These details are necessary to assess experimental reproducibility and the reliability of the reported preservation effects. The illumination conditions should also be reported, particularly because the discussion attributes part of the improved preservation to the UV-shielding properties of ZnO. An unpackaged control would provide an additional useful reference for evaluating the contribution of the packaging itself.

 

Response 9: Thank you for your suggestion. The following details were added to Section 2.5.

1. Sample quantity and replicates:

Seven blueberries were selected for each group. Three independent packaging replicates were set up (n=3). Different films (OSDF/SA, OSDF/SA/ONCC, O/CZ2, O/CZ4, O/CZ6, O/CZ8, and O/CZ10 groups) were used as sealing films. They were placed over and sealed onto the surfaces of Petri dishes containing the blueberries.

2. Storage conditions:

All samples were stored for 16 days at 20 ± 1°C and 60% relative humidity. Indicators were measured every 4 days.

3. Illumination conditions:

All samples were stored under normal indoor scattered light. Accordingly, the UV-shielding effect of ZnO in Section 3.10 was defined as an auxiliary protective mechanism.

4. Regarding the "unpackaged" control group:

Unpackaged blueberries had rapid water loss and decay rates at 20°C. This lacked a basis for parallel comparison with the coated groups. Therefore, the OSDF/SA pure matrix film group was used as the baseline control. This approach covered the basic effect of "physical coverage". It also effectively isolated and evaluated the antibacterial and structural enhancement contributions of the ONCC@ZnONP filler.

 

Comments 10: According to Table 2, O/CZ10 produces the lowest blueberry decay rate after 16 days (7.89%), compared with 15.38% for O/CZ8, whereas O/CZ8 shows better performance in terms of weight loss and firmness retention. The authors should explicitly distinguish these different outcomes and clarify the criteria used to identify the optimal formulation rather than presenting O/CZ8 as providing the best overall preservation performance without further qualification.

 

Response 10: Thank you for your suggestion. The following modifications were made.

1. Explicit distinction of results.

The O/CZ10 film had the highest ZnO loading and the strongest antibacterial efficacy. It showed the lowest decay rate (7.89%). However, this high loading caused slight ZnO agglomeration (Fig. 3). It also led to a decrease in transmittance (69.47% at 660 nm), a lower T_g, and a rebound in weight loss rate. In contrast, the O/CZ8 film achieved the optimal balance. It possessed the highest tensile strength (17.23 MPa) and the highest T_g (97.94°C). It also showed the lowest weight loss rate and the best firmness retention (9.07 N).

2. Clarification of the evaluation criteria.

The comprehensive balance between antibacterial preservation and physical quality maintenance (weight loss, firmness, and film structural integrity) was used as the criterion to determine the optimal formulation. Based on this criterion, the O/CZ8 film was identified as the formulation with the best overall preservation performance. This was not based on a single dominating indicator. This clarification was added to the Abstract, Section 3.10, and the Conclusions.

 

Comments 11: Section 3.4 refers to Figure 3B when discussing the XRD results presented in Figure 4B, while Section 3.10 incorrectly refers to Figure 5C for blueberry firmness measurements. The caption of Supplementary Figure S1 also repeats the label (D) for multiple formulations. In addition, Reference 47 does not appear appropriate for supporting the assignment of characteristic ZnO diffraction peaks. The authors should carefully verify the correspondence between citations and the associated statements, revise the figure captions, and correct the recurring grammatical errors throughout the manuscript.

 

Response 11: Thank you for the careful review and constructive comments. The entire manuscript was carefully checked, and the following modifications were made to address the above omissions.

1. In Section 3.4, the citation for the XRD results was corrected from "Fig. 3B" to "Fig. 4B". In Section 3.10, the citation for the blueberry firmness data was corrected from "Fig. 5C" to "Fig. 6C".
2. The caption of Figure S1 was revised. The repeated "(D)" labels were removed.
3. The original Reference [47] was indeed inappropriate for assigning the ZnO crystal planes. It was replaced with a reference that explicitly reported the characteristic ZnO diffraction peaks (Ashraful et al., 2025).
4. A systematic English grammar and spelling proofread of the entire manuscript was conducted. The recurring grammatical errors were corrected.

[1] Ashraful, B.A.; Mofassel, H.A.; Alam, M-A,; Alam, M-Z.; Sarker, D.; Sultana, N. Green synthesis of ZnO nanoparticles using Justicia adhatoda for effective photocatalytic degradation of methylene blue dye, RSC Adv. 2025, 15, 45874–45888.

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

1- I have a major concern regarding the use of the term "edible" to describe the developed system.< !--TgQPHd|||[]--> The formulation includes zinc oxide nanoparticles (ZnO NPs) and cellulose nanocrystals (CNC), both of which carry documented toxicity risks and are generally not cleared for human consumption. I highly recommend revising the nomenclature or re-evaluating the intended application to align with established food safety standards.

2- Add some sentences in the introduction to describe the nature, and properties of the used biopolymers. 

3- It is recommended that a two-way ANOVA be utilized for the in situ< !--TgQPHd|||[]--> analysis not one-way ANOVA.< !--TgQPHd|||[]--> This statistical approach is necessary to properly evaluate how film treatments, storage time, and the interaction between these two factors influence the monitored blueberry parameters.

Author Response

Comments 1: I have a major concern regarding the use of the term "edible" to describe the developed system. The formulation includes zinc oxide nanoparticles (ZnO NPs) and cellulose nanocrystals (CNC), both of which carry documented toxicity risks and are generally not cleared for human consumption. I highly recommend revising the nomenclature or re-evaluating the intended application to align with established food safety standards.

 

Response 1: Thank you for your suggestion. The use of the term "edible" for a system containing zinc oxide nanoparticles (ZnONPs) and cellulose nanocrystals (CNC) indeed raised major concerns. The original design of this study utilized food-grade soluble dietary fiber and sodium alginate as film-forming substrates. However, after the incorporation of nano-ZnO (even within the permitted limits for food contact materials), the primary application of this composite film was defined as active food packaging or food contact materials. It was not defined as an edible film for direct human consumption. The following modifications were made.

1. All instances of "edible packaging film" in the manuscript were replaced with "active packaging film" or "food packaging film".
2. The intended application in the Abstract and Introduction was explicitly defined as active food packaging/food contact materials. This aligned with established food safety standards.
3. The need for further verification of ZnO migration and toxicological safety was stated in the Conclusions.

 

Comments 2: Add some sentences in the introduction to describe the nature, and properties of the used biopolymers.

 

Response 2: Thank you for your suggestion. The chemical structural characteristics, film-forming properties, and crosslinking network formation mechanism of OSDF and SA were added to the second paragraph of the Introduction.

 

Comments 3: It is recommended that a two-way ANOVA be utilized for the in situ analysis not one-way ANOVA. This statistical approach is necessary to properly evaluate how film treatments, storage time, and the interaction between these two factors influence the monitored blueberry parameters.

 

Response 3: Thank you for your suggestion. The following modifications were made.

1. The statistical method for blueberry preservation indicators (decay rate, weight loss, and firmness) was changed from one-way ANOVA to two-way ANOVA. Duncan's multiple range test was retained for post-hoc multiple comparisons.
2. Statistical conclusions were added to Section 3.10. These explicitly indicated that storage time, film treatment, and their interaction had significant effects on the decay rate, weight loss, and firmness of blueberries (P < 0.05).

     3. The descriptions of the two-way ANOVA and the post-hoc test were simultaneously added to the footnote of Table 2.

Reviewer 3 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The discussion in the manuscript needs significant improvement. Particularly, mechanical properties, dsc, TGA, and antibacterial properties need to be compared with similar systems reported in the literature, and the reasons why the films developed in this paper are better or worse than similar reported systems. What are the benefits of these films compared with other polysaccharide composite films containing zinc oxide and/or cellulose in any nanoform? Furthermore, there are numerous papers published regardng biobased coatings on blueberries. How the films developed in this manuscript are better or worse in comparison with other coatings for blueberries? For how much % they prolong the shelf-life of blueberries according to assesed blueberries parameters in comparison to reported biobased coatings?

Author Response

Comments 1: The discussion in the manuscript needs significant improvement. Particularly, mechanical properties, dsc, TGA, and antibacterial properties need to be compared with similar systems reported in the literature, and the reasons why the films developed in this paper are better or worse than similar reported systems. What are the benefits of these films compared with other polysaccharide composite films containing zinc oxide and/or cellulose in any nanoform? Furthermore, there are numerous papers published regardng biobased coatings on blueberries. How the films developed in this manuscript are better or worse in comparison with other coatings for blueberries? For how much % they prolong the shelf-life of blueberries according to assesed blueberries parameters in comparison to reported biobased coatings?

 

Response 1: Thank you for your suggestion.

1. The discussion section of the revised manuscript systematically compared the developed films with similar reported systems. This comparison was conducted from two aspects: interfacial structure design and various physicochemical properties. Unlike conventional polysaccharide/ZnO composite films prepared by physical blending, the core advantage of the film developed in this manuscript lay in the in situ hydrothermal growth strategy. This strategy used ONCC as a biotemplate to anchor ZnO in situ, constructing a continuous and dense organic-inorganic dual-functional interfacial network. This structure effectively inhibited nanofiller agglomeration and improved interfacial compatibility. Consequently, it synergistically enhanced the mechanical properties, thermal stability, and antibacterial performance of the composite films.

① Mechanical properties: Films of agar, carrageenan, and CMC directly doped with ZnO showed significantly decreased tensile strength due to weak interfacial interactions (Kanmani et al., 2014). In contrast, ONCC@ZnONP achieved efficient stress transfer and simultaneous enhancement of tensile strength by constructing a dense interfacial network.

② DSC (thermal transition): The simple blending strategy easily led to nano-ZnO agglomeration and weakened interfacial interactions, providing limited restriction on polymer chain segmental motion (Oun et al., 2020). However, ONCC@ZnONP significantly restricted OSDF chain activity through dense interfacial bonding. This endowed the composite films with a higher apparent T_g.

③ TGA (thermal stability): The maximum thermal degradation temperature (400°C) of the physically doped ZnO/phycocyanin sago starch system was lower than that of pure starch (412°C) (Erami et al., 2025). In contrast, ONCC@ZnONP effectively delayed the thermal cracking of the main chain through a dense interface. It achieved a higher apparent T_50% while increasing the char yield, demonstrating better thermal stability.

④ Antibacterial properties: In situ loaded ZnO/cellulose nanocomposites exhibited enhanced antibacterial activity due to improved particle dispersion (Lefatshe et al., 2017). Based on the same in situ growth mechanism, the O/CZ10 film also exhibited prominent inhibition zones against Escherichia coli and Staphylococcus aureus.

2. Regarding the comparison of blueberry preservation effects, Section 3.10 of the revised manuscript provided a quantitative comparison with reported biobased coatings. Compared with other blueberry biobased coatings, the film developed in this paper exhibited superior long-term preservation efficacy. Based on the decay rate as a key parameter for evaluating fruit commercial value, a decay rate below 10% was used as the effective shelf-life endComments. The chitosan biobased coating maintained blueberry quality for approximately 12 days at room temperature (Duan et al., 2011). In contrast, the O/CZ10 film extended the time to 16 days for maintaining equivalent quality. This represented a shelf-life extension of approximately 33%, demonstrating superior long-term preservation efficacy.

[1] Kanmani, P.; Rhim, J-W. Properties and characterization of bionanocomposite films prepared with various biopolymers and ZnO nanoparticles. Carbohyd Polym, 2014, 106, 190-199.

[2] Oun, A. A., Shankar, S., Rhim, J.W. Multifunctional nanocellulose/metal and metal oxide nanoparticle hybrid nanomaterials. Crit Rev Food Sci, 2020,60(3), 435–460.

[3] Erami, F.S.; Hosseinzadeh, M.; Zardoost, M.R.; Khalilzadeh, M.A. Physicochemical properties of biodegradable edible films developed from sago starch: effects of phycocyanin, nano-ZnO, and a combination of experiments and theory. Int. J. Biol. Macromol, 2025, 318, 144947.

[4] Lefatshe, K.; Muiva, C-M.; Kebaabetswe, L-P. Extraction of nanocellulose and in-situ casting of ZnO/cellulose nanocomposite with enhanced photocatalytic and antibacterial activity. Carbohyd Polym, 2017, 164, 301-308.

[5] Duan, J.Y.; Wu, R.Y.; Strik,B-C.; Zhao, Y.Y. Effect of edible coatings on the quality of fresh blueberries (Duke and Elliott) under commercial storage conditions. Postharvest Biol Tec, 2011, 59, 71-79.

Round 2

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The authors have satisfactorily addressed my concerns. No additional modifications are needed.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Major revision:

a) novelty should be better explained in introduction. There are numerous papers published on alginate/ZnO system, and alginate/ZNO/cellulose fiber system, and are not cited, neither is sufficiently elaborated why this work is advantegous in comparison to the published papers. 

b) figure 3 should be improved, resolution is poor, and should contain less images

c) the results are well presented but not discussed at all. Authors should include discussion related to mechanical, optical, antibacterial results and blueberries decay, why they obtain the trends they obtained, and to compare with literature their mechanical, optical, antibacterial properties, and coating efficiency on blueberries.

Author Response

Comments 1: The novelty should be better explained in introduction. There are numerous papers published on alginate/ZnO system, and alginate/ZNO/cellulose fiber system, and are not cited, neither is sufficiently elaborated why this work is advantegous in comparison to the published papers.

Response 1: Thank you.  In the Introduction of the manuscript, a substantial number of relevant references on sodium alginate/ZnO and sodium alginate/ZnO/cellulose composite films have been added, and the innovations and advantages of the present study compared with previous studies have been clarified. Specifically, this study innovatively used orange peel pomace soluble dietary fiber (OSDF) as the main film-forming matrix, with sodium alginate (SA) serving only as a co-film-forming agent, thereby achieving high-value utilization of citrus processing waste. Meanwhile, ONCC@ZnONP was synthesized in situ using orange peel pomace cellulose nanocrystals (ONCC) as a biological template, integrating the mechanical reinforcement of ONCC with the antibacterial and UV-shielding functions of ZnO within a single hybrid unit and effectively avoiding the agglomeration issues associated with the direct addition of ZnO alone. Therefore, this work was superior to conventional single-component fillers or simple blending systems in terms of both the source of the film matrix and the integrated design of filler functionality.

 Comments 2: Figure 3 should be improved, resolution is poor, and should contain less images.

 Response 2: Thank you for your suggestion. The surface and cross-section SEM images of the O/CZ2 and O/CZ6 films in Fig.3 have been removed, and the overall resolution of the images has been improved.

Comments 3: The results are well presented but not discussed at all. Authors should include discussion related to mechanical, optical, antibacterial results and blueberries decay, why they obtain the trends they obtained, and to compare with literature their mechanical, optical, antibacterial properties, and coating efficiency on blueberries.

Response 3: Thank you. Section 3.10 of the manuscript was substantially expanded by adding in-depth discussion on the mechanical, optical, and antibacterial properties as well as the decay behavior of blueberries. The underlying mechanisms behind the observed trends were explained, and references reporting similar findings were added and cited.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

1- Title: the SA should be included in the title to be (Edible Films Based on Orange Peel Pomace Soluble Dietary Fiber and sodium alginate Reinforced with Dual-Functional Cellulose Nanocrystal/Zinc Oxide Hybrids for Antibacterial Food Packaging).

2- Keywords length can be shortened. 

 3- Introduction can be improved by mentioning the limitations of using edible biopolymers-based active packaging.

4-   Materials and reagents: a- properties of the used polymers (SA) and chemicals should be specified (ex: M. wt, viscosity...etc).

b- Bacteria names should be italicized throughout the whole manuscript. 

c- what are the properties of the prepared ONCC (crystallinity, size ..etc).

5- Barrier properties of films such as Water vapor permeability and oxygen transmission rate should be performed. 

6- DSC method is not clear. the authors should clearly indicate the conditions (cycles numbers , type of pan used..)? the DSC graph from the first or the second cycle ? how the Tg was calculated ??

7- For the antimicrobial evaluation, the range of bacterial concentration used (10^4 -10^8) is very wide range of 4 log difference which is not acceptable. the range difference should at maximum be 0.5 log. 

8- for the bacterial count experiment, the results should be presented in terms of CFU/mL, not just photos.

9- Discussion can be better improved by linking the different characterization studies such as the SEM, DSC, XRD, FTIR, Mechanical properties. 

10- I suggest mention the limitation of the study in the conclusion section. 

Comments on the Quality of English Language

I suggest reconsider after major revision.

Author Response

Comments 1: Title, the SA should be included in the title to be (Edible Films Based on Orange Peel Pomace Soluble Dietary Fiber and sodium alginate Reinforced with Dual-Functional Cellulose Nanocrystal/Zinc Oxide Hybrids for Antibacterial Food Packaging).

Response 1: Thank you for your suggestion. The manuscript title has been revised to “Edible Films Based on Orange Peel Pomace Soluble Dietary Fiber and Sodium Alginate Reinforced with Bifunctional Cellulose Nanocrystal/Zinc Oxide Hybrids for Antimicrobial Food Packaging”. Sodium alginate (SA) has been incorporated into the title to accurately reflect the matrix composition of the edible films.

 Comments 2: Keywords length can be shortened.

Response 2: Thank you for your suggestion. The keywords have been shortened by retaining the core and distinctive keywords, making them more concise and standardized in accordance with the journal's keyword guidelines.

Comments 3: Introduction can be improved by mentioning the limitations of using edible biopolymers-based active packaging.

Response 3: Thank you. The introduction has been improved by mentioning the limitations of using edible biopolymers-based active packaging.

Comments 4: Materials and reagents: a- properties of the used polymers (SA) and chemicals should be specified (ex: M. wt, viscosity...etc). b- Bacteria names should be italicized throughout the whole manuscript. c- what are the properties of the prepared ONCC (crystallinity, size.etc).

Response 4: Thank you. The properties of the used polymers, chemicals, and ONCC have been supplemented in the Materials and Reagents, and the bacteria names have been italicized throughout the whole manuscript.

Comments 5: Barrier properties of films such as Water vapor permeability and oxygen transmission rate should be performed.

Response 5: Thank you for your suggestion. Barrier properties, such as water vapor permeability and oxygen transmission rate, are indeed among the important evaluation indicators for edible packaging films. However, the core objective of the present study was to develop an antibacterial edible film using orange peel pomace soluble dietary fiber (OSDF) and sodium alginate as the matrix, reinforced with a bifunctional cellulose nanocrystal/zinc oxide hybrid (ONCC@ZnONP), and to systematically evaluate its structural, mechanical, UV-shielding, and antibacterial properties as well as its practical application performance in blueberry preservation.

The blueberry preservation experiments have provided indirect but compelling evidence for the improved water vapor barrier property of the O/CZ films. As shown in Fig. 6B and Table 2, after 16 days of storage, the weight loss rate of blueberries packaged with the O/CZ8 film was only 9.67%, compared with 14.72% for the OSDF/SA control group. Since weight loss during storage is primarily attributed to moisture evaporation through the packaging film, this significant reduction directly reflects the enhanced water vapor barrier property of the O/CZ films. Moreover, the decay rate of the O/CZ10 film (7.89%) was much lower than that of the OSDF/SA film (72.35%), indicating that the film effectively inhibited the growth of aerobic spoilage microorganisms, which is partly attributed to the improved oxygen barrier performance. Meanwhile, it has been reported in the literature that in similar polysaccharide-based film systems, the incorporation of cellulose nanocrystals and zinc oxide nanoparticles generally reduces water vapor permeability and oxygen permeability, owing to the increased tortuosity of the diffusion path and the formation of a denser network structure [1-6]. Based on these findings, the preservation results and the relevant literature support the conclusion that the barrier properties of the O/CZ films are improved. In future work, systematic measurements of water vapor permeability and oxygen transmission rate are planned as part of a more comprehensive performance evaluation.

[1] Rahman, S.; Batsh, C.; Gurumayam, S.; Borah, J.C.; Chowdhury, D. Sodium alginate-nanocellulose-based active composite film for edible oils packaging applications, Materials Advances. 2024, 5, 9314-9329.

[2] Yang, J.; Saqib, M.N.; Liu, F.; Zhong, F. Bacterial cellulose nanocrystals with a great difference in aspect ratios: a comparison study of their reinforcing effects on properties of the sodium alginate film, Food Hydrocoll. 2023, 141, 108676.

[3] S. Abdel Aziz, M.; E. Salama, H. Development of alginate-based edible coatings of optimized UV-barrier properties by response surface methodology for food packaging applications, Int. J. Biol. Macromol. 2022, 212, 294–302.

[4] Motelica, L.; Ficai, D.; Oprea, O.; Ficai, A.; Trusca, R.; Andronescu, D.E.; Holban, A.M. Biodegradable alginate films with ZnO nanoparticles and citronella essential oil—a novel antimicrobial structure, Pharmaceutics. 2021, 13, 1020.

[5] Motelica, L.; Ficai, D.; Oprea, O.C.; Ficai, A.; Ene, V.L.; Vasile, B.S.; Andronescu, E.; Holban, A.M. Antibacterial biodegradable films based on alginate with silver nanoparticles and lemongrass essential oil-innovative packaging for cheese, Nanomaterials-basel. 2021, 11, 2377.

[6] Oun, A.A.; Rhim, J.W. Carrageenan-based hydrogels and films: effect of ZnO and CuO nanoparticles on the physical, mechanical, and antimicrobial properties, Food Hydrocoll. 2017, 67, 45–53.

Comments 6: DSC method is not clear. the authors should clearly indicate the conditions (cycles numbers , type of pan used..)? the DSC graph from the first or the second cycle ? how the Tg was calculated ??.

Response 6: Thank you. In Section 2.4.4 of the manuscript, the DSC method (single heating cycle, aluminum crucible) and the method for determining the glass transition temperature (T_g) were supplemented. The DSC curves presented were obtained from the first heating cycle. The T_g values were determined using the equal-area method specified in ISO 11357-2:2020, corresponding to the extrapolated onset temperature (T_onset).

Comments 7: For the antimicrobial evaluation, the range of bacterial concentration used (10^4 -10^8) is very wide range of 4 log difference which is not acceptable. the range difference should at maximum be 0.5 log.

Response 7: Thank you. After checking the original experimental records, it was confirmed that the manuscript contained an inaccurate description. The concentration range of 1 × 10⁴ to 1 × 10⁸ CFU/mL referred only to the serially diluted bacterial suspensions used in the preliminary screening experiments, and this wide range spanning four log units was not used in the formal antibacterial experiments. All formal antibacterial experiments were conducted using a uniform fixed bacterial concentration of 1 × 10⁶ CFU/mL. The corresponding description in the manuscript was revised accordingly.

Comments 8: for the bacterial count experiment, the results should be presented in terms of CFU/mL, not just photos.

Response 8: Thank you for your suggestion. In the plate counting experiments of this study, the OSDF/SA and OSDF/SA/ONCC films showed excessive colony growth that exceeded the countable range (>300 CFU/plate). Therefore, accurate CFU/mL values and inhibition rates could not be obtained.

Accordingly, the estimated inhibition rate values were removed from the manuscript. The inhibition zone diameter was adopted as the quantitative indicator of antibacterial activity, supplemented by plate photographs as visual evidence, which clearly reflected the dose-dependent antibacterial effect of the O/CZ films.

Antibacterial performance was not the core objective of this study but served only as a supplementary evaluation of the edible multifunctional films. In future studies, the dilution scheme will be optimized to obtain reliable CFU/mL data for further quantitative analysis.

Comments 9: Discussion can be better improved by linking the different characterization studies such as the SEM, DSC, XRD, FTIR, Mechanical properties.

Response 9: Thank you for your suggestion. The Discussion has been supplemented and improved by establishing a correlation analysis among the multiple characterization results of the films, thereby enriching the discussion on the structure–property mechanisms.

Comments 10: I suggest mention the limitation of the study in the conclusion section.

Response 10: Thank you for your suggestion. The limitations of the study and future research perspectives have been supplemented in the Conclusions section.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have considered my comments in the revised version of the manuscript. I have no outstanding comments anymore than to recommend the paper for publication acceptance.