Myotube/Adipocyte Powder-Enriched Alginate–Zein Hydrogels Support Myotube Alignment for 3D Myoblast Culture

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1.Lines 21-22: The full form of an abbreviation (FTIR, SEM etc.) should be written the first time it appears in the Abstract.

2.Line 32: The first letter of each keyword should be capitalized.

3. Line 36: What are the advantages of biomaterials as scaffolds compared to other materials? Such comparisons should be mentioned in the introduction.

4.Lines 62-64: Someexamples should be given composed by zein and Algi, and what’s their advantages compared to other scaffolds?

5.Lines 99-102: The primary research methods employed, and their corresponding research objectives should be mentioned.

6.The logic of the entire Introduction section is not sufficiently clear. Please refine the description and clarify the relationships.

7.Lines 149-151: Passive voice should be used to convey objectivity.

8.Lines 183-185: Refine descriptions and avoid excessive use of the sameconjunctions.

9.Line226: The full form of PBST should be written the first time it appears.

10.The results （mean±SD） in the entire text are rounded to two decimal places.

11.Please provide a detailed explanation of the data analysis methods. What were the significance analyses for Figures 1a, 1b, and 2c? Who is being compared with? Didn't understand the meaning of the annotations.

12.The results and discussion suggest that this should be compared and analyzed with the research on cell-cultured meat scaffolds in other reference.

Author Response

We sincerely thank the reviewers for their insightful and constructive comments. We have carefully revised the manuscript accordingly. All revisions are highlighted in red in the revised manuscript.

Reviewer 1

Comment 1

Lines 21–22: The full form of an abbreviation (FTIR, SEM etc.) should be written the first time it appears in the Abstract.

Response 1:

All abbreviations (e.g., FTIR and SEM) have been defined in full at their first occurrence in the Abstract.

 

Comment 2

Line 32: The first letter of each keyword should be capitalized.

Response 2:

The keywords have been revised so that the first letter of each keyword is capitalized (Abstract, Lines 27–28). “Keywords: Alginate; Adipocytes; C2C12; Cell powder; Cell culture; Cultured meat; Cell alignment; Zein”

Comment 3

Line 36: What are the advantages of biomaterials as scaffolds compared to other materials? Such comparisons should be mentioned in the introduction.

Response 3:

A comparative statement has been added to the Introduction describing the advantages of biomaterial-based scaffolds over synthetic materials, including cytocompatibility, biodegradability, ECM-mimicking properties, and suitability for food-grade applications.

• Introduction: Lines (41-52): “Biomaterial-based scaffolds have garnered increasing attention in cultured-meat and tissue-engineering applications due to their intrinsic biocompatibility, biodegradability, and ability to mimic the native ECM microenvironment [9]. Synthetic polymeric scaffolds often require surface modification to support cell attachment and may generate non-degradable residues. Naturally derived biomaterials provide inherent cell-adhesive motifs, facilitate nutrient diffusion, and degrade into biologically tolerated by-products [10]. In the context of cultured-meat production, food-grade biomaterials further offer advantages in regulatory acceptance, edibility, and consumer safety [3, 11]. Moreover, biomaterial scaffolds can recapitulate the biochemical and mechanical cues of muscle ECM, thereby supporting myoblast alignment, fusion, and maturation more effectively than inert synthetic matrices [12, 13]. These features make biomaterial-based scaffolds particularly suitable for engineering structured, edible muscle tissues for cultivated-meat applications.”

 

Comment 4

Lines 62–64: Some examples should be given composed by zein and Algi, and what’s their advantages compared to other scaffolds?

Response 4

Representative literature examples of composite scaffolds incorporating alginate or zein with other biomaterials (e.g., gelatin, collagen, and chitosan) have been added to the Introduction.

• Introduction: Lines (73-80): ‘’Previous studies have shown that incorporating zein into polysaccharide- or protein-based scaffolds markedly improves their suitability for muscle tissue engineering. For example, zein–chitosan and zein–gelatin composites exhibited enhanced mechanical strength and significantly improved C2C12 cell adhesion and alignment compared with chitosan- or gelatin-only [23-25]. Similarly, incorporation of Algi into collagen or gelatin based hydrogels improved water retention, nutrient diffusion, and cytocompatibility, resulting in higher cell viability and differentiation compared to single collagen or gelatin-matrices [26, 27].’’

 

Comment 5

Lines 99–102: The primary research methods employed, and their corresponding research objectives should be mentioned.

Response 5:

This section has been revised to explicitly state the primary experimental approaches (scaffold fabrication, physicochemical characterization, and biological evaluation) and their corresponding research objectives.

• Introduction: Lines (97-110) : “Despite recent advances in edible scaffold development, several critical limitations remain. First, most food-grade scaffolds primarily provide mechanical support and lack lineage-specific biochemical bioactivity capable of simultaneously delivering myogenic and adipogenic cues within a single matrix [44]. Second, the induction of myotube alignment typically relies on external patterning strategies, molds, or mechanical stimulation, which limits fabrication simplicity and scalability [45]. Third, many alignment-directing scaffolds lack comprehensive food-grade biochemical vali-dation, restricting their translational relevance to cultured-meat applications [44, 45]. Collectively, these limitations underscore the need for edible scaffold systems that integrate intrinsic bioactivity, scalable self-alignment capacity, and food-grade bio-chemical functionality. Accordingly, the primary objective of this study was to evaluate whether the in-corporation of MP/AP into Algi/zein hydrogels enhances myoblast alignment com-pared with MP/AP-free scaffolds. As a secondary objective, we further assessed whether MP/AP-derived cues confer muscle-like biochemical characteristics.’’

 

Comment 6

The logic of the entire Introduction section is not sufficiently clear. Please refine the description and clarify the relationships.

Response 6:

The Introduction has been revised to improve logical flow, clarify the relationships between prior studies and the research gap, and strengthen the rationale for the present study.

• Introduction: Lines (97-114) : “Despite recent advances in edible scaffold development, several critical limitations remain. First, most food-grade scaffolds primarily provide mechanical support and lack lineage-specific biochemical bioactivity capable of simultaneously delivering myogenic and adipogenic cues within a single matrix [44]. Second, the induction of myotube alignment typically relies on external patterning strategies, molds, or mechanical stimulation, which limits fabrication simplicity and scalability [45]. Third, many alignment-directing scaffolds lack comprehensive food-grade biochemical vali-dation, restricting their translational relevance to cultured-meat applications [44, 45]. Collectively, these limitations underscore the need for edible scaffold systems that integrate intrinsic bioactivity, scalable self-alignment capacity, and food-grade bio-chemical functionality. Accordingly, the primary objective of this study was to evaluate whether the in-corporation of MP/AP into Algi/zein hydrogels enhances myoblast alignment com-pared with MP/AP-free scaffolds. As a secondary objective, we further assessed whether MP/AP-derived cues confer muscle-like biochemical characteristics. We hypothesized that incorporation of MP and AP powders into Algi/zein hydrogels would provide intrinsic biochemical and topographical cues sufficient to enhance myotube alignment without the need for external patterning, while also introducing muscle-like biochemical features.’’

 

Comment 7

Lines 149–151: Passive voice should be used to convey objectivity.

Response 7:

The indicated sentences have been revised to use passive voice to enhance objectivity.

• Methods: Lines (162-164): “Finally, MP and AP were prepared from differentiated C2C12 myotubes and adipocytes, respectively, and used as powdered supplements for subsequent scaffold composition and cell culture experiments.’’

 

Comment 8

Lines 183–185: Refine descriptions and avoid excessive use of the same conjunctions.

Response 8:

The indicated text has been revised to improve clarity, reduce repetitive conjunctions, and enhance readability

-  Methods: (Lines 193-196): “In the 3D experiments, AP and MP powders were mixed at a 1:1 ratio and incorporated into the Algi/zein hydrogel (Algi/zein (AP: MP)). A 1 mL aliquot of the hydrogel was transferred into a 4-well culture plate and combined with 5 × 10⁴ C2C12 cells. The constructs were subsequently crosslinked using 2% CaCl₂ in distilled water for 5 min.’’

 

Comment 9

Line 226: The full form of PBST should be written the first time it appears.

Response 9:

The full term “phosphate-buffered saline with Tween-20 (PBST)” has been provided at its first occurrence.

• Methods: Lines (243-244): “phosphate-buffered saline with Tween-20 (PBST).’’

 

Comment 10

Results (mean ± SD) should be rounded to two decimal places.

Response 10:

All reported mean ± SD values have been rounded to two decimal places throughout the manuscript.

Comment 11

Please provide a detailed explanation of the data analysis methods. What were the significance analyses for Figures 1a, 1b, and 2c? Who is being compared with? Didn't understand the meaning of the annotations.

Response 11:

The statistical analysis methods and figure annotations have been clarified in the revised manuscript. Swelling ratios of Algi and Algi/zein hydrogels were measured repeatedly over a 10-day culture period (Figure 1a). Because the analysis included two independent variables (scaffold type and time) with repeated measurements, the data was analyzed using a two-way repeated-measures ANOVA. When occasionally missing data points occurred, a mixed-effects model (REML) was applied. Post hoc pairwise comparisons between Algi and Algi/zein at each time point were performed using the Holm–Šidák multiple-comparisons test. Statistical significance indicates differences between scaffold types at the same time point. Asterisks denote significance levels (**p < 0.01, ***p < 0.001, ****p < 0.0001).

 

For biodegradation, no statistically significant differences were detected between Algi and Algi/zein at any time point (Figure 1b). The absence of asterisks indicates that no statistically significant difference was observed. Figure 2c was deleted during manuscript modification.

- Methods: Lines (286-293): “All quantitative data are presented as mean ± standard error (SE). Statistical analyses were performed using GraphPad Prism version 8.0.1.244 (64-bit, Windows). Data were analyzed using two-way ANOVA with repeated measures. When missing data points were present, mixed-effects models (REML) were applied. Post hoc comparisons were corrected using Holm–Šidák’s multiple-comparison tests, as appropriate. For each condition, n represents the number of individual hydrogel samples (scaffold replicates) analyzed per group (n = 4–8), as indicated in the figure legends. Adjusted p-values (q-values) < 0.05 were considered statistically significant.’’

- Figure 1 legend: Lines (335-337): “Data were analyzed using a two-way ANOVA, followed by Šidák corrected post-hoc comparisons between scaffolds at each time point. Significance was denoted by **p < 0.01, ***p < 0.001, and ****p < 0.0001.’’

 

Comment 12

The results and discussion suggest that this should be compared and analyzed with the research on cell-cultured meat scaffolds in other reference.

Response 12:

The Discussion has been expanded to include comparisons with recent cultured meat scaffold studies, highlighting similarities, differences, and the specific contributions of the present work.

• Discussion: Lines (592-615): ’’In the context of cultured meat development, several edible scaffold systems have been reported, including Algi, gelatin, soy-protein, and plant-derived porous scaffolds designed to support myoblast attachment and differentiation. Algi and gelatin-based scaffolds promote cell viability and alignment but primarily provide physical support with limited tissue-specific biochemical signaling [27]. Soy protein and plant-based scaffolds offer food compatibility but often lack muscle or adipose-specific biological cues [70]. Compared with these reported systems, the present Algi/zein (AP: MP) scaffold integrates both food-grade structural support and lineage-specific biochemical com-ponents derived from muscle and adipose tissues, enabling enhanced cellular alignment and partial biochemical mimicry. In addition, several contemporary cultured meat approaches rely on lay-er-by-layer or compartmentalized scaffold architectures in which adipocyte and myocyte-laden matrices are physically assembled. However, these constructions often re-main biochemically segregated, thereby restricting reciprocal biochemical signaling between muscle and adipose components. For example, gelatin and soymilk-based scaffolds have been used to fabricate fat-containing cultured meat by stacking individually differentiated muscle and adipose-like layers forming composite tissues. However, these layers remain biochemically distinct rather than fully integrated within a single matrix [44]. Edible 3D Algi–gelatin hydrogel scaffolds have also been developed to support cell growth in structured cultured meat models. However, these systems primarily provide mechanical support without integrated lineage-specific biochemical cues [71]. In contrast, the present Algi/zein (AP: MP) composite scaffold integrates muscle and adipose-derived biochemical cues within a single edible matrix, enabling concurrent structural support and lineage-specific bioactivity without the need for live co-culture systems.’’

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, below are some considerations with my suggestions for improving the work.

 Limitations of the study: please revise and include in the paper the limitations in the discussion and conclusion of the results (if not performed during the study include this as a limitation)

The manuscript lacks testable hypotheses and predefined primary/secondary outcomes (e.g., a priori thresholds for “improved alignment” or “enhanced differentiation”). This makes interpretation vulnerable to bias. Please add explicit hypotheses and define primary endpoints

Controls are incomplete, comparisons omit key materials controls (e.g., zein-only, Algi-only with AP:MP, and Algi/zein without CaCl₂ crosslinking), and species-relevant controls (murine muscle tissue) given that C2C12 is a mouse line. Include these controls to isolate material effects and to avoid conflating powder bioactivity with alginate/zein synergy.

The functional readouts are absent, the study infers maturation from morphology/immunostaining but lacks functional assays (contractility, calcium transients, or electrical stimulation responses).

Food-relevant validation is limited: sensory, rheology (texture), digestibility, and safety (residual solvents, endotoxin/bioburden) are not assessed, yet the scaffold is proposed as “edible”.

Time-course viability (days 1–10) and multi-group comparisons (scaffold × time; AP:MP ratios) are analyzed with one‑way ANOVA, which does not account for repeated measures and factorial structure. Use two‑way ANOVA with repeated measures (time × scaffold), mixed-effects models when missing points, and report post-hoc corrections (Tukey/Holm–Šidák). Provide effect sizes (η² or Cohen’s d), exact p-values, and 95% CIs.

Figures list n = 8, but methods state different sampling and disk punching; clarify whether n denotes biological replicates (independent cultures) or technical replicates (disks from one culture).

Numerous gene markers and time-points are compared without a correction strategy—control the false discovery rate across gene panels.

You attribute alignment in 3D “without external molds”, but earlier use a food mold in 2D optimization; ensure conclusions on spontaneous alignment are based exclusively on mold‑free experiments and that 3D alignment is quantified vs. Algi/zein alone.

Zein is dissolved in 75% ethanol then “removed” by stirring; later, metabolites show ethanol as a feature (Table 3), risking interpretation as a scaffold trait rather than residual solvent.

AP dissolved in DMSO (0.1%). DMSO can alter cell behaviour?

Abstract. overgeneralized conclusion (“effective approach… closely mimic real meat”) despite non‑significant gene upregulation in Figure 8d. Please state explicit quantitative gains (e.g., % increase in 0° orientation peak), (ii) temper claims to match the non‑significant gene data

Introduction. please add sharper gap statements: (a) need for integrated lineage‑specific bioactivity, (b) scalable, edible alignment cues without patterns/molds, and (c) food‑grade validation. End with a clear hypothesis and specific aims.

Conclusion: to vague. Also, overstates “enhanced resemblance to beef” while Table 3 shows mixed signals and putative identifications. Revise to bounded claims and list future work. Rewrite the conclusion to emphasize alignment improvements and cytocompatibility as demonstrated, acknowledge non‑significant gene changes, and propose a clear future work plan (functional contractility, MSI-compliant metabolomics, rheology/texture, sensory). Frame translational value as potential rather than achieved.

2.3.4 SEM magnifications: “1,000 K X, 10,000 K X, and 20,000 K X” likely means 1,000×, 10,000×, 20,000×; “K” implies 1,000× multiplier (i.e., 1,000,000×), which is unrealistic for SEM

2.3.7 qPCR: Using RNeasy Plant Mini Kit for mammalian cells is atypical and may bias yield/integrity; switch to a mammalian RNA kit, report RIN, primer efficiencies, reference gene validation, and adhere to MIQE (checklist in SI). Correct abbreviation in Abbreviations

2.3.3 Physical characteristics: The swelling ratio description suggests W₀ is obtained after freeze-drying at each time point; clarify whether W₀ is baseline dry mass pre‑swelling or time‑specific dry mass post‑incubation, and use standard SR = (W_t–W_dry)/W_dry ×100 with separate samples for swelling and degradation to avoid circularity

FTIR: Report instrument resolution realistically (0.06 cm⁻¹ is unusually fine for routine FTIR), number of scans, ATR vs. transmission, and perform baseline correction. Provide deconvoluted amide I analysis (secondary structure fractions) if claiming protein structural features.

Metabolomics: Specify identification confidence (MSI levels), reference libraries (e.g., NIST/Wiley), match scores, and whether identifications are putative vs. confirmed. Current practice requires clear levels to avoid overinterpretation.

Results 3.7.3 Heatmap paragraph: The text interprets RT peaks as specific metabolites (acetaldehyde, ethanol, hexanal) without reporting m/z, fragment ions, or library match scores

Morphological alignment and myosin immunostaining are compelling; however, qPCR of late markers (MyoG, Myh1, Myoz1, fusion markers) shows non‑significant enhancement in Algi/zein AP:MP (Fig. 8d), contradicting the narrative of “enhanced differentiation”. Temper claims and consider power analysis and additional markers (e.g., TnT, MHC isoforms) to reconcile morphology with gene expression.

Interpretation suggests “completion of proliferation phase”; alternatively, scaffold nutrient diffusion limits or degradation dynamics may reduce viability. Add diffusion modelling/rheology or media perfusion to support the mechanistic explanation

The presence of ethanol likely stems from processing; hexanal differences indicate oxidative profiles diverge from beef. Conclusions should state “partial chemical similarity” and specify the features driving clustering, with MSI levels and QC described.

The work demonstrates feasible alignment and cytocompatibility of food‑grade composites—important for cultured meat scaffolding—but without functional muscle performance, robust molecular confirmation, and food-grade validations, the claim that these scaffolds “closely mimic real meat” is premature.

The review covers alginate/zein scaffolds and cultured meat broadly, but could better integrate recent edible scaffold scale‑up and food safety/regulatory discussions (e.g., annual reviews and gel-based hybrid strategies). Add coverage of scalable edible scaffolds and rheological demands.

Figure 8d vs. text. Text claims enhanced differentiation, but gene upregulation is non‑significant—align the narrative to the data

GraphPad Prism version. Methods say “GraphPad Prism 8” and “version 10.2.0”—resolve this discrepancy and specify OS, analysis modules, and multiple‑comparison tests.

Terminology. Standardize “cultured meat” (not “culture meat” in keywords) and fix “Myomarker” → Myomaker in abbreviations.

Table 3 (GC–MS analysis). RT values like “RT‑15365” seem to be typos

Identification is putative without m/z or MS/MS—label as MSI Level 2/3; move interpretive aroma notes to SI unless confirmed with standards.

Distinguish process artifacts (ethanol from zein prep) from biological signals with proper controls.

Author Response

We sincerely thank the reviewers for their insightful and constructive comments. We have carefully revised the manuscript accordingly. All revisions are highlighted in red in the revised manuscript.

Reviewer 2

Comment 1

Limitations of the study: please revise and include in the paper the limitations in the discussion and conclusion of the results (if not performed during the study include this as a limitation)

Response 1:

The limitations of the study have been explicitly stated in both the Discussion and Conclusion sections, including the use of a murine cell model, the absence of functional muscle assays, and the lack of food-grade validation and qPCR analysis.

-Discussion: Lines (656-674): “Despite these advances, several limitations should be acknowledged. This study was conducted using the murine C2C12 myoblast cell line, which may not fully recapitulate the behavior of livestock-derived muscle cells relevant to commercial cultured meat production. Importantly, functional muscle performance, including contractility, calcium transients, or responses to electrical stimulation, was not evaluated; therefore, no conclusions regarding functional maturation were drawn. Muscle differentiation was inferred solely from morphological alignment and myosin immunostaining. Although trends were observed, the lack of qPCR analysis under the present experimental conditions indicates the need for larger sample sizes and additional markers (e.g., TnT, MHC isoforms) in future studies. In addition, food-relevant properties such as texture, rheology, diffusion characteristics, media perfusion, sensory attributes, and long-term stability were not assessed. While metabolomic profiling suggested partial biochemical similarity to native beef tissue, compound identification remained putative, limiting chemical interpretation. These limitations highlight the need for further validation using livestock-derived cells, functional assays, and comprehensive food-grade evaluations. Finally, the absence of mechanical stimulation, long-term maturation analysis, and in vivo validation, together with the use of murine rather than primary bovine myoblasts, may limit the direct translational scalability of this platform to industrial cultured meat production.’’

-Conclusion: Lines (679-684): “Metabolomic profiles showed partial overlap with beef; however, all metabolite identifications were putative (MSI Level 3). Overall, these findings support the potential of MP/AP-enriched Algi/zein hydrogels as a food-grade scaffold platform for structured cultured meat applications, rather than demonstrating chemical equivalence to native beef. Further studies employing MSI-compliant metabolomic validation and advanced quality control will be required to strengthen translational relevance.”

 

Comment 2

The manuscript lacks testable hypotheses and predefined primary/secondary outcomes (e.g., a priori thresholds for “improved alignment” or “enhanced differentiation”). This makes interpretation vulnerable to bias. Please add explicit hypotheses and define primary endpoints

Response 2:

Explicit hypotheses and predefined endpoints have been added to the manuscript.

 The primary hypothesis was that incorporating MP and AP into Algi/zein hydrogels enhances myoblast alignment compared with MP/AP-free Algi/zein control scaffolds. The primary endpoint was the quantitative myotube alignment analysis. In addition, the study evaluated whether MP/AP incorporation introduces biochemical features associated with native muscle tissue as a secondary outcome. These secondary analyses are presented as supportive evidence, and their limitations are explicitly acknowledged in the revised manuscript.

• Introduction: Lines (107-114): “Accordingly, the primary objective of this study was to evaluate whether the in-corporation of MP/AP into Algi/zein hydrogels enhances myoblast alignment compared with MP/AP-free scaffolds. As a secondary objective, we further assessed whether MP/AP derived cues confer muscle-like biochemical characteristics. We hypothesized that incorporation of MP and AP powders into Algi/zein hydrogels would provide intrinsic biochemical and topographical cues sufficient to enhance myotube alignment without the need for external patterning, while also introducing muscle-like biochemical features.”
• Conclusion: Lines (676-680): “This study demonstrates that MP/AP-enriched Algi/zein hydrogels enable robust intrinsic myotube alignment without external molds while maintaining cytocompatible growth of C2C12 myoblasts, representing the primary functional outcome of the scaffold system. Metabolomic profiles showed partial overlap with beef; however, all metabolite identifications were putative (MSI Level 3).“

 

 

Comment 3

Controls are incomplete, comparisons omit key materials controls (e.g., zein-only, Algi-only with AP:MP, and Algi/zein without CaCl₂ crosslinking), and species-relevant controls (murine muscle tissue) given that C2C12 is a mouse line. Include these controls to isolate material effects and to avoid conflating powder bioactivity with alginate/zein synergy.

Response 3:

 This point is acknowledged as a limitation. Additional material and species-relevant control groups could further isolate the individual contributions of the powders and the alginate/zein matrix. However, fabrication of zein-only hydrogel is technically challenging because zein is hydrophobic and does not readily form a stable hydrogel without incorporation into a polysaccharide matrix and an appropriate crosslinking process. In addition, CaCl₂ is required for ionic crosslinking of alginate; without CaCl₂, a stable gel cannot be formed or maintained during extended culture.

 

 The CaCl₂ concentration used in this study was within a non-cytotoxic range; therefore, the direct effect of crosslinking on cellular outcomes was considered minimal. To assess powder-related effects while maintaining scaffold integrity, comparisons were performed using MP/AP-free Algi/zein controls and multiple AP:MP ratio conditions. Nonetheless, the absence of the additional proposed control groups is recognized as a limitation and has been explicitly stated in the revised Discussion section.

• Discussion: Lines (640-644): “Although the present study establishes proof-of-concept, inclusion of additional material- and species-specific control groups and extended functional validation would provide further mechanistic insight; however, such controls were technically constrained by the physicochemical requirements for stable Algi/zein hydrogel formation and will be addressed in future studies.”

 

Comment 4

The functional readouts are absent, the study infers maturation from morphology/immunostaining but lacks functional assays (contractility, calcium transients, or electrical stimulation responses).

Response 4:

Functional readouts such as contractility, calcium transients, and responses to electrical stimulation were not assessed in this study. Therefore, muscle maturation was evaluated only using morphological observations and immunostaining, and no conclusions were drawn regarding functional maturation. This limitation has been explicitly stated in the Discussion, and incorporation of functional assays is identified as an important direction for future studies.

• Discussion: Lines (656-661): “Despite these advances, several limitations should be acknowledged. This study was conducted using the murine C2C12 myoblast cell line, which may not fully recapitulate the behavior of livestock-derived muscle cells relevant to commercial cultured meat production. Importantly, functional muscle performance, including contractility, calcium transients, or responses to electrical stimulation, was not evaluated; therefore, no conclusions regarding functional maturation were drawn.”

 

Comment 5

Food-relevant validation is limited: sensory, rheology (texture), digestibility, and safety (residual solvents, endotoxin/bioburden) are not assessed, yet the scaffold is proposed as “edible”.

Response 5:

Food-relevant validation was not performed in this study. Specifically, sensory attributes, rheological properties (texture), digestibility, and safety-related assessments (e.g., residual solvents, endotoxin/bioburden, and microbial load) were not evaluated. Accordingly, although the scaffold is intended for edible applications, additional food-specific testing is required before edibility-related claims can be confirmed. This limitation has been clarified in the Discussion, and these analyses are proposed as important future work.

• Discussion: Lines (665-670): “In addition, food-relevant properties such as texture, rheology, diffusion characteristics, media perfusion, sensory attributes, and long-term stability were not assessed. While metabolomic profiling suggested partial biochemical similarity to native beef tissue, compound identification remained putative, limiting chemical interpretation. These limitations highlight the need for further validation using livestock-derived cells, functional assays, and comprehensive food-grade evaluations.”

 

Comment 6

Time-course viability (days 1–10) and multi-group comparisons (scaffold × time; AP:MP ratios) are analyzed with one‑way ANOVA, which does not account for repeated measures and factorial structure. Use two‑way ANOVA with repeated measures (time × scaffold), mixed-effects models when missing points, and report post-hoc corrections (Tukey/Holm–Šidák). Provide effect sizes (η² or Cohen’s d), exact p-values, and 95% CIs.

Response 6:

The AP:MP ratio data across time were reanalyzed using a two-way repeated-measures ANOVA (time × group). When occasional missing time points were present, a mixed-effects model (REML) was applied. Post hoc multiple comparisons were performed using the Holm–Šidák correction, and the specific statistical approach is now reported in the Methods and corresponding figure legends. This reanalysis altered the statistical outcomes for certain comparisons, and all affected results and interpretations have been updated accordingly in the revised manuscript.

For all revised analyses, effect sizes (η²), exact p-values, and 95% confidence intervals are now reported (Table below).

• Methods: Lines (286-293): “All quantitative data are presented as mean ± standard error (SE). Statistical analyses were performed using GraphPad Prism version 8.0.1.244 (64-bit, Windows). Data was analyzed using two-way ANOVA with repeated measures. When missing data points were present, mixed-effects models (REML) were applied. Post hoc comparisons were corrected using Holm–Šidák’s multiple-comparison tests, as appropriate. For each condition, n represents the number of individual hydrogel samples (scaffold replicates) analyzed per group (n = 4–8), as indicated in the figure legends. Adjusted p-values (q-values) < 0.05 were considered statistically significant.”
• Figure 1 legend: Lines (335-337): “Data was analyzed using a two-way ANOVA, followed by Šidák corrected post-hoc comparisons between scaffolds at each time point. Significance was denoted by **p < 0.01, ***p < 0.001, and ****p < 0.0001.”
• Figure S1 Legend: “Data was analyzed using a two-way ANOVA with MP concentration and time as factors, followed by Šidák-corrected post-hoc comparisons between concentrations at each time point. Significance was denoted ****P < 0.00012.”
• Table 1a-c (below)

 

Table 1a. Pairwise post-hoc comparisons (Šidák-adjusted) for swelling and biodegradability of Algi vs. Algi/zein scaffolds over time. (Figure 1a&b)

Swelling (Time point)	P value	95% CIs	Significance
D1	0.0001	942.6 to 1929	Yes
D3	0.0002	821.1 to 2174	Yes
D5	0.0031	685.3 to 2173	Yes
D7	0.0001	1387 to 2024	Yes
D10	0.0001	1232 to 2042	Yes
Biodegradability (Time point)	P value	95% CIs	Significance
D1	0.4550	-57.26 to 19.07	No
D3	0.9763	-14.25 to 10.50	No
D5	0.9988	-10.72 to 8.99	No
D7	0.9639	-19.60 to 29.30	No
D10	0.9997	-21.21 to 18.24	No

 

For swelling, two-way ANOVA showed a significant main effect of scaffold type (p < 0.0001, η² = 0.34), whereas the main effect of time was not significant (p = 0.3844, η² = 0.035), and the scaffold × time interaction was not significant (p = 0.6590, η² = 0.021). Šidák-corrected post hoc comparisons indicated significantly greater swelling in Algi compared with Algi/zein at D1, D3, D5, D7, and D10.

 

For biodegradation, two-way ANOVA revealed a significant main effect of time (p = 0.0021, η² = 0.11), whereas the main effect of scaffold type was not significant (p = 0.2758, η² = 0.09), and the scaffold × time interaction was not significant (p = 0.8878). Šidák-corrected post hoc comparisons showed no significant differences between Algi and Algi/zein at any individual time point.

Table 1b. Outer and inner surface pore characteristics of Algi and Algi/zein hydrogels (SEM analysis) (Figure 1d-e& g-h)

 

Outer surface	P value	95% CIs	Significance
Pore area (%)	0.2591	-4.49 to 16.20	No
Average pore size (μm)	0.999	-0.193 to 0.193	No
Inner surface	P value	95% CIs	Significance
Pore area (%)	0.3459	-7.29 to 3.23	No
Average pore size (μm)	0.2532	-11.23 to 3.24	No

 

Two-way ANOVA of the outer surface showed no significant main effect of scaffold type on pore characteristics (p = 0.1306, η² = 0.018), and no significant scaffold × parameter interaction was detected (p = 0.6822, η² = 0.029). Šidák-corrected post hoc comparisons confirmed no significant differences between Algi and Algi/zein scaffolds for either pore area or average pore size.

Two-way ANOVA of the inner surface showed no significant main effect of scaffold type on pore characteristics (p = 0.2746, η² = 0.10), and no significant scaffold × parameter interaction was detected (p = 0.6077, η² = 0.05). Šidák-corrected post hoc comparisons confirmed no significant differences between Algi and Algi/zein scaffolds for inner-surface pore area or average pore size.

Table 1c. Time-dependent effects of MP concentration on C2C12 cell viability (CCK-8 assay) (Figure 4c)

Time point	Comparison	P value	95% CIs	Significance
D1	Control vs 0.01μg/μL	0.9999	-8.31 to 7.45	No
	Control vs 0.05μg/μL	0.1709	-17.64 to 3.77	No
	Control vs 0.1μg/μL	0.0760	-26.20 to -1.94	No
D3	Control vs 0.01μg/μL	0.0377	0.97 to 30.26	Yes
	Control vs 0.05μg/μL	0.0157	1.68 to 13.64	Yes
	Control vs 0.1μg/μL	0.999	-90.50 to 957	No
D5	Control vs 0.01μg/μL	0.5292	-10.80 to 25.93	No
	Control vs 0.05μg/μL	0.7161	-18.48 to 35.13	No
	Control vs 0.1μg/μL	0.3822	-28.18 to 9.33	No

 

Time-course viability data were analyzed using REML with MP concentration. The analysis revealed a significant effect on day3 (η² = 0.29, p < 0.0001), whereas the main effect of MP concentration was not significant (η² = 0.06, p = 0.054), and no significant MP concentration × time interaction (η² = 0.03, p = 0.088). Post hoc Šidák-corrected comparisons were reported.

 

Comment 7

Figures list n = 8, but methods state different sampling and disk punching; clarify whether n denotes biological replicates (independent cultures) or technical replicates (disks from one culture).

Response 7:

 The definition of n has been clarified in the revised manuscript. In this study, n represents the number of individual hydrogel samples (scaffold replicates) analyzed per condition (typically n = 4–8).

• Methods : Lines (286-293): “All quantitative data are presented as mean ± standard error (SE). Statistical analyses were performed using GraphPad Prism version 8.0.1.244 (64-bit, Windows). Data was analyzed using two-way ANOVA with repeated measures. When missing data points were present, mixed-effects models (REML) were applied. Post hoc comparisons were corrected using Holm–Šidák’s multiple-comparison tests, as appropriate. For each condition, n represents the number of individual hydrogel samples (scaffold replicates) analyzed per group (n = 4–8), as indicated in the figure legends. Adjusted p-values (q-values) < 0.05 were considered statistically significant”

Comment 8

Numerous gene markers and time-points are compared without a correction strategy—control the false discovery rate across gene panels.

Response 8:

 False discovery rate (FDR) control was applied to the multi-gene comparisons using the Benjamini–Hochberg procedure. For each experimental time point, overall ANOVA p-values were extracted and adjusted for multiple testing. After correction, none of the tested genes remained statistically significant (q > 0.05). Accordingly, to avoid overinterpretation, the gene expression results were removed from the main manuscript, and this limitation has been stated in the revised discussion.

 

Comment 9

You attribute alignment in 3D “without external molds”, but earlier use a food mold in 2D optimization; ensure conclusions on spontaneous alignment are based exclusively on mold‑free experiments and that 3D alignment is quantified vs. Algi/zein alone.

Response 9:

All 3D experiments reported in the revised manuscript were conducted without external molds or patterning devices. Myotube alignment in 3D was quantified in MP/AP-enriched Algi/zein scaffolds and directly compared with MP/AP-free Algi/zein controls. The previously included food-grade mold experiment (Submitted manuscript) was part of an early optimization step and has been fully removed to avoid confusion. The Introduction, Discussion, and Conclusions have been revised to ensure that all statements regarding spontaneous alignment are based exclusively on mold-free 3D experiments and on the direct comparison against MP/AP-free Algi/zein scaffolds.

• Introduction: Lines (111-114): “We hypothesized that incorporation of MP and AP powders into Algi/zein hydrogels would provide intrinsic biochemical and topographical cues sufficient to enhance myotube alignment without the need for external patterning, while also introducing muscle-like biochemical features.”
• Discussion: Lines (661-662):“Muscle differentiation was inferred solely from morphological alignment and myosin immunostaining.”
• Conclusion: Lines (676-678): “This study demonstrates that MP/AP-enriched Algi/zein hydrogels enable robust intrinsic myotube alignment without external molds while maintaining cytocompatible growth of C2C12 myoblasts, representing the primary functional outcome of the scaffold system. “

 

Comment 10

Zein is dissolved in 75% ethanol then “removed” by stirring; later, metabolites show ethanol as a feature (Table 3), risking interpretation as a scaffold trait rather than residual solvent.

Response 10:

 Ethanol was used only as a temporary solvent to dissolve zein during scaffold preparation and was not intended to be a structural or functional component of the scaffold. Following zein dissolution, ethanol was removed through repeated stirring and solvent exchange steps prior to cell culture and downstream analyses.

Accordingly, the ethanol peak detected in the GC–MS analysis should not be interpreted as an intrinsic scaffold-related metabolite. Any residual ethanol signal likely reflects sample handling and/or extraction-related background rather than solvent retained within the scaffold during culture. The Methods section has been revised to clarify the role and removal of ethanol during fabrication, and the interpretation of Table S1 has been updated to prevent misattribution of ethanol-related signals to scaffold composition.

• Methods: Lines (171-172): “Ethanol was used only as a transient solvent and was removed before analysis. The homogenate of Algi/zein hydrogel was stirred using a magnetic bar at 50°C.”
• Table S1 interpretation: Red color interpretation: “Detected in all samples. Ethanol was used only as a transient solvent during zein dissolution and was removed prior to cell culture and GC–MS analysis. Therefore, ethanol signals should not be interpreted as intrinsic scaffold-derived metabolites and likely reflect background or sample-processing-related traces.”

 

Comment 11

AP dissolved in DMSO (0.1%). DMSO can alter cell behaviour?

Response 11:

AP was dissolved in DMSO because it did not dissolve directly in culture medium, resulting in a final DMSO concentration of 0.1% (v/v). This concentration is commonly used in cell culture and is generally considered non-cytotoxic (Reference 68). In the present study, Live/Dead staining, the CCK-8 assay, and PAX7 immunofluorescence (Figure 5a–b) showed no adverse effects under the tested conditions, indicating that 0.1% DMSO did not measurably alter cell viability or behavior in this system. This clarification has been added to the revised manuscript.

• Methods: Lines (190-192): “While AP was dissolved in 0.1% Dimethyl sulfoxide (DMSO) (Sigma Aldrich) before preparing AP: MP ratios. The final DMSO concentration in all culture conditions did not exceed 0.1% (v/v).”
• Discussion: Lines (587-590): “Although AP was dissolved in 0.1% DMSO for preparation purposes, this low concentration is widely tolerated in in vitro systems [68] and did not interfere with cytocompatibility or myogenic differentiation under the present experimental conditions.”
• Reference (68) Figure 1A-C

 

Comment 12

Abstract. overgeneralized conclusion (“effective approach… closely mimic real meat”) despite non significant gene upregulation in Figure 8d. Please state explicit quantitative gains (e.g., % increase in 0° orientation peak), (ii) temper claims to match the non significant gene data

Response 12:

The Abstract has been revised to remove overgeneralized statements and to align conclusions with the reported data. Desmin-positive myotube orientation was quantified across −90° to 90°, with the proportion of axially aligned fibers (0° ± 10°) used as the primary alignment metric. MP/AP incorporation increased axial alignment, with the 1:1 AP:MP condition yielding the highest proportion of aligned myotubes. The Abstract and Results now report these quantitative alignment improvements explicitly. In addition, because gene expression changes were not statistically significant, the gene expression findings were removed from the main manuscript and are acknowledged as a limitation in the Discussion.

• Abstract: Lines (20-23): “MP/AP incorporation generated ECM-like microstructures and significantly enhanced myotube alignment in Algi/zein scaffolds compared with MP/AP-free controls, in-creasing the proportion of axially aligned fibers by up to ~6-fold at a 1:1 AP: MP ratio.”
• Results: Lines (429-440): “Immunostaining for desmin was used to visualize cytoskeletal organization and myo-genic differentiation. MP: AP -treated cells (1:1, 2:1, and 4:1) exhibited more uniform desmin organization compared with control (1:0), whereas a higher MP: AP ratio (6:1) was associated with disrupted desmin integrity (Figure 5d). Myotube alignment (des-min⁺ myotubes) was analyzed from −90° to 90°, and the fusion index at 0° was used as the primary orientation endpoint. All MP/AP-enriched media showed a general alignment shift toward ~30° angle. Among these, the 1:1 AP: MP formulation exhibited the greatest enrichment of axially aligned fibers (0° ± 10°). Specifically, the 1:0, 1:1, 2:1, 4:1, and 6:1 AP: MP groups showed 125%, 617%, 136%, 512%, and 169% , respectively (Figure 5e). Our findings indicate that the AP: MP 1:1 ratio represented the viability of C2C12 cells and supported their proliferation and differentiation with a prospective effect of ~ 6-fold myotube alignment.”
• Discussion: Lines (662-664): “Although trends were observed, the lack of qPCR analysis under the present experimental conditions indicates the need for larger sample sizes and additional markers (e.g., TnT, MHC isoforms) in future studies.”

 

Comment 13

Introduction. please add sharper gap statements: (a) need for integrated lineage specific bioactivity, (b) scalable, edible alignment cues without patterns/molds, and (c) food grade validation. End with a clear hypothesis and specific aims.

Response 13:

The Introduction has been revised to more clearly articulate the key research gaps, including the need for lineage-specific bioactivity, scalable edible approaches to promote myogenic organization without external molds/patterning, and the current limitations in food-grade validation. The revised Introduction now concludes with an explicit hypothesis and clearly defined study aims.

• The need for integrated lineage-specific bioactivity: Lines (97-100): “Despite recent advances in edible scaffold development, several critical limitations remain. First, most food-grade scaffolds primarily provide mechanical support and lack lineage-specific biochemical bioactivity capable of simultaneously delivering myogenic and adipogenic cues within a single matrix [44].”
• Scalable, edible alignment cues without patterns/molds : Lines (100-102) : “Second, the induction of myotube alignment typically relies on external patterning strategies, molds, or mechanical stimulation, which limits fabrication simplicity and scalability [45].”
• Food grade validation : Lines (102-106) : “Third, many alignment-directing scaffolds lack comprehensive food-grade biochemical validation, restricting their translational relevance to cultured-meat applications [44, 45]. Collectively, these limitations underscore the need for edible scaffold systems that integrate intrinsic bioactivity, scalable self-alignment capacity, and food-grade bio-chemical functionality.”
• The hypothesis has been clarified : Lines (111-114): “We hypothesized that incorporation of MP and AP powders into Algi/zein hydrogels would provide intrinsic biochemical and topographical cues sufficient to enhance myotube alignment without the need for external patterning, while also introducing muscle-like biochemical features.”

Comment 14

Conclusion: to vague. Also, overstates “enhanced resemblance to beef” while Table 3 shows mixed signals and putative identifications. Revise to bounded claims and list future work. Rewrite the conclusion to emphasize alignment improvements and cytocompatibility as demonstrated, acknowledge non significant gene changes, and propose a clear future work plan (functional contractility, MSI-compliant metabolomics, rheology/texture, sensory). Frame translational value as potential rather than achieved.

Response 14:

The Conclusion has been rewritten to ensure that all claims are appropriately bound and directly supported by the data. The revised text emphasizes the demonstrated outcomes of improved myotube alignment and cytocompatibility and describes the metabolomics results as putative with mixed directional trends rather than definitive resemblance to beef. In addition, a clear future work plan has been added, including functional contractility assessment, MSI-compliant metabolomics, rheological/texture characterization, and sensory evaluation. The translational relevance is now framed as potential rather than achieved.

• Conclusion: Lines (676-684): “This study demonstrates that MP/AP-enriched Algi/zein hydrogels enable robust intrinsic myotube alignment without external molds while maintaining cytocompatible growth of C2C12 myoblasts, representing the primary functional outcome of the scaffold system. Metabolomic profiles showed partial overlap with beef; however, all metabolite identifications were putative (MSI Level 3) . Overall, these findings support the potential of MP/AP-enriched Algi/zein hydrogels as a food-grade scaffold platform for structured cultured meat applications, rather than demonstrating chemical equivalence to native beef. Further studies employing MSI-compliant metabolomic validation and advanced quality control will be required to strengthen translational relevance.”

 

Comment 15

2.3.4 SEM magnifications: “1,000 K X, 10,000 K X, and 20,000 K X” likely means 1,000×, 10,000×, 20,000×; “K” implies 1,000× multiplier (i.e., 1,000,000×), which is unrealistic for SEM

Response 15:

SEM magnifications have been corrected

• Methods: Lines (222-224): “Images captured at 10,000× magnification were used to quantify the pore area percentage and average pore size using ImageJ software (version 1.47).”

 

Comment 16

2.3.7 qPCR: Using RNeasy Plant Mini Kit for mammalian cells is atypical and may bias yield/integrity; switch to a mammalian RNA kit, report RIN, primer efficiencies, reference gene validation, and adhere to MIQE (checklist in SI). Correct abbreviation in Abbreviations

Response 16:

 In the current 3D culture system, RNA was extracted from cell-seeded plant-derived scaffolds. During preliminary optimization, the use of a conventional mammalian RNA extraction kit resulted in insufficient RNA recovery and inconsistent yield due to the dense scaffold matrix and interference from scaffold-derived components. Therefore, the RNeasy Plant Mini Kit was used for scaffold-derived samples because it includes a homogenization step that improves disruption of the scaffold material and enables more reliable RNA extraction from these constructs.

 

In response to the reviewer’s request, qPCR experiments were repeated using a 2D C2C12 proliferation model, and RNA was extracted using a standard mammalian RNA extraction kit. Primer efficiencies were re-evaluated, and the gene expression data were analyzed using the statistical approach requested by the reviewer, including correction for multiple testing where applicable. However, the repeated analysis did not yield statistically significant gene expression differences. Accordingly, to avoid overinterpretation, the qPCR results were removed from the manuscript in their entirety, and this limitation has been stated in the revised Discussion.

• Discussion: Lines (Lines 662–665): “Although trends were observed, the lack of qPCR analysis under the present experimental conditions indicates the need for larger sample sizes and additional markers (e.g., TnT, MHC isoforms) in future studies.”
• RNA concentration and purity were assessed using a Tecan Infinite 200 with a NanoQuant plate (A260/280), which provides spectrophotometric quality metrics but does not generate RNA integrity numbers (RIN).
• GAPDH showed stable Ct values across conditions (Proliferation: 19.57 ± 1.2, differentiation: 19.66 ± 0.57), supporting its use as a reference gene. Primer performance was further confirmed by a standard curve (R² = 0.982, efficiency = 118.83%).
• Primer efficiencies were determined using serially diluted cDNA and are reported as: P21 (99.9%, R² = 0.999), Cdk1 (111.9%, R² = 0.998), MyoG (125.9%, R² = 0.960), Desmin (119.9%, R² = 0.962), and MHC3 (122.6%, R² = 0.971).

 

Comment 17

2.3.3 Physical characteristics: The swelling ratio description suggests W₀ is obtained after freeze-drying at each time point; clarify whether W₀ is baseline dry mass pre swelling or time specific dry mass post incubation, and use standard SR = (W_t–W_dry)/W_dry ×100 with separate samples for swelling and degradation to avoid circularity

Response 17:

 

W₀ represents the baseline dry mass of each scaffold measured prior to swelling and was not re-measured after incubation. In addition, independent scaffold samples were used for each time point to avoid circularity between swelling and degradation measurements.

• Methods: Lines (202-209): “Briefly, the swelling ratio (SR) of hydrogels with a uniform thickness of 7 mm was evaluated at 37 °C and 5% CO₂. Before incubation, each scaffold was freeze-dried at −50 °C for 24 h, and its initial dry mass (W0) was recorded as the baseline reference weight. Hydrogels were then incubated in fresh culture medium for 0 (immediately post-gelation), 1, 3, 5, 7, and 10 days, with medium replaced every two days. At each point, independent samples were gently blotted to remove excess surface liquid, and their wet mass (Wt) was recorded. The swelling ratio was calculated using the standard formula: ??(%)= ??−?0/?0×100 [47].”

 

Comment 18

FTIR: Report instrument resolution realistically (0.06 cm⁻¹ is unusually fine for routine FTIR), number of scans, ATR vs. transmission, and perform baseline correction. Provide deconvoluted amide I analysis (secondary structure fractions) if claiming protein structural features.

Response 18:

 FTIR acquisition and reporting details have been revised for clarity and accuracy. Spectra were collected in ATR mode using a Cary 670 bench spectrometer coupled with a Cary 620 microscope. Although the instrument has an optical resolution capability of >0.06 cm⁻¹, all spectra in this study were acquired at a spectral resolution of 4 cm⁻¹. For each sample, 32 scans were recorded and averaged. Baseline correction and peak deconvolution were not performed.

 Accordingly, the manuscript has been revised to remove any claims regarding quantitative protein secondary structure. The observed bands in the amide I (~1600–1700 cm⁻¹) and amide II (~1510–1580 cm⁻¹) regions are reported only as qualitative evidence of protein-associated functional groups, consistent with prior literature (Reference 49). These methodological details and corresponding revisions have been incorporated into the updated Methods and Results sections.

• Table 1: Line (481 in red color): “Confirms protein presence from the MP component and partial retention in the scaffold. no secondary structure analysis performed [49].”
• Methods: Lines (256-264): “FTIR spectroscopy of Algi/zein and Algi/zein (AP: MP) hydrogel scaffolds were performed using a Cary 670 bench spectrometer coupled with a Cary 620 microscope (Agilent Technologies, Santa Clara, CA, USA). Samples were measured in ATR mode over the range 500–4500 cm⁻¹ with a spectral resolution of 4 cm⁻¹, and 32 scans per sample were recorded (number of scans averaged for each spectrum: 32). Hydrogels were measured directly without additional pre-treatment. Spectra were plotted as transmittance versus wavenumber using GraphPad Prism software. No baseline correction or Amide I deconvolution was applied. Observed peaks were interpreted based on standard band assignments for proteins, lipids, and carbohydrates [49].”
• Results: Lines (456-467): “FTIR spectroscopy was employed to characterize the chemical features and compositional differences of Algi/zein and Algi/zein (AP: MP) hydrogels (Figure 6). All spectra showed broad absorption bands around 3270–3300 cm⁻¹, indicating O–H and N–H stretching vibrations associated with proteins and water. The MP spectrum displayed prominent peaks at ~1650 cm⁻¹ (amide I, C=O stretching) and ~1540 cm⁻¹ (amide II, N–H bending), consistent with protein presence [49]. In contrast, the AP spectrum showed weaker amide bands but more pronounced absorptions in the region of 1200–2000 cm⁻¹, likely associated with C–O stretching of carbohydrates and lipid-related groups. The control sample (Algi/zein) exhibited lower overall intensity and fewer distinct absorption peaks. These spectral features reflect the unique molecular compositions of the Algi/zein (AP: MP) hydrogel. MP is protein-dominant, whereas AP shows lipid and carbohydrate signatures (Table 1).”

 

Comment 19

Metabolomics: Specify identification confidence (MSI levels), reference libraries (e.g., NIST/Wiley), match scores, and whether identifications are putative vs. confirmed. Current practice requires clear levels to avoid overinterpretation.

Response 19:

Metabolite identities were assigned based on previously published retention times (RT) and mass spectral data (references provided in Table S1), rather than by comparison to spectral libraries (e.g., NIST/Wiley) or authentic standards. Therefore, all metabolite identifications are reported as putative and correspond to MSI Level 3. The Methods and Results sections have been revised to explicitly state the identification confidence level and to avoid overinterpretation of these assignments.

• Methods: Lines (282-283): “Identifications were reported at MSI Level 3. Hierarchical clustering heatmaps were used for data visualization.”
• Results: Lines (540-544): “All peak assignments are putative (MSI Level 3) based on previously published retention times (Table S1), without confirmation by authentic standards. These results suggest that incorporation of AP and MP components alters the volatile metabolite profile of the scaffold toward patterns that partially overlap with beef, based on putative RT-based assignments.”
• Table S1 note: ”All metabolite identifications are putative (MSI Level 3) and were assigned based on previously reported retention times. No authentic standards, MS/MS fragmentation, or library match scores were obtained.”

 

Comment 20

Results 3.7.3 Heatmap paragraph: The text interprets RT peaks as specific metabolites (acetaldehyde, ethanol, hexanal) without reporting m/z, fragment ions, or library match scores

Response 20:

In the revised manuscript, the heatmap results are described based on retention times and clustering patterns without assigning specific metabolite identities. All compound annotations are reported as putative (MSI Level 3) based on previously published RT and mass spectral data (Reference 36 and references listed in Table S1), without authentic standards or library match scores.

• Methods: Lines (282-283): “Identifications were reported at MSI Level 3. Hierarchical clustering heatmaps were used for data visualization.”
• Results: Lines (530-544): “A hierarchical clustering heatmap was generated to compare metabolite profiles among beef, composite Algi/zein, and Algi/zein (AP:MP) scaffolds. The retention time (RT) values displayed distinct clustering patterns, indicating putative differences in metabolite composition across the groups (Figure 8). The Algi/zein (AP:MP) scaffold exhibited stronger signals at RT-1.296 and RT-1.076, approaching those of beef, suggesting the putative presence of metabolites associated with muscle tissue. In contrast, the Algi/zein scaffold showed moderate signals at RT-1.296 and RT-3.7042, with minimal activity at RT-1.076. Beef samples clustered separately, exhibiting a broader range of RT intensities, including high signals at RT-7.925 and RT-1.296, reflecting their native metabolic complexity. All peak assignments are putative (MSI Level 3) based on previously published retention times (Table S1), without confirmation by authentic standards. These results suggest that incorporation of AP and MP components alters the volatile metabolite profile of the scaffold toward patterns that partially overlap with beef, based on putative RT-based assignments.”

 

 

Comment 21

Morphological alignment and myosin immunostaining are compelling; however, qPCR of late markers (MyoG, Myh1, Myoz1, fusion markers) shows non significant enhancement in Algi/zein AP:MP (Fig. 8d), contradicting the narrative of “enhanced differentiation”. Temper claims and consider power analysis and additional markers (e.g., TnT, MHC isoforms) to reconcile morphology with gene expression.

Response 21:

The gene expression data did not show statistically significant upregulation. Accordingly, the manuscript has been revised to ensure that conclusions are aligned with the revised data. The primary demonstrated outcome is now reported as improved myotube alignment and morphological organization.

• Abstract: Lines (25-26): “Incorporating MP and AP into Algi/zein hydrogels enhanced myotube alignment and showed partial structural and biochemical similarity to native muscle tissue.”
• Results : Lines (437-440): “Our findings indicate that the AP: MP 1:1 ratio represented the viability of C2C12 cells and supported their proliferation and differentiation with a prospective effect of ~ 6-fold myotube alignment.”
• Discussion: Lines (661-665): “Muscle differentiation was inferred solely from morphological alignment and myosin immunostaining. Although trends were observed, the lack of qPCR analysis under the present experimental conditions indicates the need for larger sample sizes and additional markers (e.g., TnT, MHC isoforms) in future studies.”
• Conclusion: Lines (676-678): “This study demonstrates that MP/AP-enriched Algi/zein hydrogels enable robust intrinsic myotube alignment without external molds while maintaining cytocompatible growth of C2C12 myoblasts, representing the primary functional outcome of the scaffold system.”

 

Comment 22

Interpretation suggests “completion of proliferation phase”; alternatively, scaffold nutrient diffusion limits or degradation dynamics may reduce viability. Add diffusion modelling/rheology or media perfusion to support the mechanistic explanation

Response 22:

The interpretation has been revised to acknowledge that nutrient diffusion limitations and scaffold degradation dynamics may contribute to the observed viability trends, in addition to proliferation-related effects. Accordingly, the revised manuscript discusses these outcomes as a combination of biological and scaffold-dependent factors rather than attributing them solely to completion of the proliferation phase. Diffusion modeling, rheological characterization, and perfusion-based culture are identified as important future studies to further clarify the underlying mechanisms.

• Discussion Lines (645-655): “Recent studies have highlighted that edible scaffolds for cultured meat should not only support cellular functions but also meet requirements for scalable manufacturing, food safety compliance, and tailored rheological properties[79]. Hybrid gel-based scaffold systems, composed of naturally derived polymers such as alginate, plant proteins, and polysaccharides with food-grade functional additives, have been proposed as a promising platform that balances biocompatibility, edibility, mechanical integrity, and consumer safety, facilitating scalable production approaches such as extrusion or printing [79, 80]. These hybrid materials enhance mechanical stability and rheological behavior, which are critical for maintaining structural integrity during culture and downstream processing, and align with regulatory frameworks for edible components (e.g., GRAS status) [81].”

 

• Discussion: Lines (565-574): “In addition, food-relevant properties such as texture, rheology, diffusion characteristics, media perfusion, sensory attributes, and long-term stability were not assessed. While metabolomic profiling suggested partial biochemical similarity to native beef tissue, compound identification remained putative, limiting chemical interpretation. These limitations highlight the need for further validation using livestock-derived cells, functional assays, and comprehensive food-grade evaluations. Finally, the absence of mechanical stimulation, long-term maturation analysis, and in vivo validation, together with the use of murine rather than primary bovine myoblasts, may limit the direct translational scalability of this platform to industrial cultured meat production.”

 

Comment 23

The presence of ethanol likely stems from processing; hexanal differences indicate oxidative profiles diverge from beef. Conclusions should state “partial chemical similarity” and specify the features driving clustering, with MSI levels and QC described.

Response 23:

The Conclusions have been revised to describe the metabolomics findings as indicating partial chemical similarity rather than equivalence to beef. In addition, the interpretation of ethanol-related signals has been updated to reflect likely processing- or handling-related origins. The revised text also specifies that clustering is driven by retention time–based features and reports metabolite annotations as putative (MSI Level 3), with appropriate clarification to avoid overinterpretation.

• Conclusion: Line (676-684): “This study demonstrates that MP/AP-enriched Algi/zein hydrogels enable robust intrinsic myotube alignment without external molds while maintaining cytocompatible growth of C2C12 myoblasts, representing the primary functional outcome of the scaffold system. Metabolomic profiles showed partial overlap with beef; however, all metabolite identifications were putative (MSI Level 3). Overall, these findings support the potential of MP/AP-enriched Algi/zein hydrogels as a food-grade scaffold platform for structured cultured meat applications, rather than demonstrating chemical equivalence to native beef. Further studies employing MSI-compliant metabolomic validation and advanced quality control will be required to strengthen translational relevance.”
• Table S1

 

Comment 24

The work demonstrates feasible alignment and cytocompatibility of food grade composites—important for cultured meat scaffolding but without functional muscle performance, robust molecular confirmation, and food-grade validations, the claim that these scaffolds “closely mimic real meat” is premature.

Response 24:

The manuscript has been revised to avoid overstated claims. In the absence of functional muscle performance assays, robust molecular confirmation, and comprehensive food-grade validation, the scaffold is no longer described as closely mimicking native meat. Instead, the revised text frames the Algi/zein MP/AP system as a food-grade scaffold that partially recapitulates selected structural and biochemical features relevant to cultured meat applications.

• Abstract: Line (25-26): “Incorporating MP and AP into Algi/zein hydrogels enhanced myotube alignment and showed partial structural and biochemical similarity to native muscle tissue.”
• Results: Line (540-544): “All peak assignments are putative (MSI Level 3) based on previously published retention times (Table S1), without confirmation by authentic standards. These results suggest that incorporation of AP and MP components alters the volatile metabolite profile of the scaffold toward patterns that partially overlap with beef, based on putative RT-based assignments.”
• Discussion: Lines (559-561): “ This study demonstrates that incorporating MP and AP powders into Algi/zein hydrogels produces edible scaffolds that partially mimic the structural and biochemical features of muscle tissue.”
• Discussion: Lines (667-669): “While metabolomic profiling suggested partial biochemical similarity to native beef tissue, compound identification remained putative, limiting chemical interpretation.”
• Conclusion: Lines (679-680): “Metabolomic profiles showed partial overlap with beef; however, all metabolite identifications were putative (MSI Level 3).”

 

Comment 25

The review covers alginate/zein scaffolds and cultured meat broadly, but could better integrate recent edible scaffold scale up and food safety/regulatory discussions (e.g., annual reviews and gel-based hybrid strategies). Add coverage of scalable edible scaffolds and rheological demands.

Response 25:

 The Discussion has been expanded to better integrate recent literature on scalable edible scaffold development, including gel-based hybrid strategies and food safety/regulatory considerations. In addition, rheological requirements relevant to scaffold performance and potential scale-up are now discussed to strengthen the translational context.

• Discussion: Lines: (645-655): “Recent studies have highlighted that edible scaffolds for cultured meat should not only support cellular functions but also meet requirements for scalable manufacturing, food safety compliance, and tailored rheological properties[79]. Hybrid gel-based scaffold systems, composed of naturally derived polymers such as alginate, plant proteins, and polysaccharides with food-grade functional additives, have been proposed as a promising platform that balances biocompatibility, edibility, mechanical integrity, and consumer safety, facilitating scalable production approaches such as extrusion or printing [79, 80]. These hybrid materials enhance mechanical stability and rheological behavior, which are critical for maintaining structural integrity during culture and downstream processing, and align with regulatory frameworks for edible components (e.g., GRAS status) [81].”
• Discussion; Lines: (665-667): “In addition, food-relevant properties such as texture, rheology, diffusion characteristics, media perfusion, sensory attributes, and long-term stability were not assessed.”
• Discussion: Lines (592-601): “In the context of cultured meat development, several edible scaffold systems have been reported, including Algi, gelatin, soy-protein, and plant-derived porous scaffolds designed to support myoblast attachment and differentiation. Algi and gelatin-based scaffolds promote cell viability and alignment but primarily provide physical support with limited tissue-specific biochemical signaling [27]. Soy protein and plant-based scaffolds offer food compatibility but often lack muscle or adipose-specific biological cues [70]. Compared with these reported systems, the present Algi/zein (AP: MP) scaffold integrates both food-grade structural support and lineage-specific biochemical com-ponents derived from muscle and adipose tissues, enabling enhanced cellular alignment and partial biochemical mimicry.“

 

 

Comment 26

Figure 8d vs. text. Text claims enhanced differentiation, but gene upregulation is non significant—align the narrative to the data

Response 26:

Statements implying enhanced differentiation have been removed because the gene expression changes were not statistically significant. The revised manuscript now aligns the narrative with the data by emphasizing the demonstrated structural and alignment outcomes rather than transcriptional upregulation.

• Results: Lines (508-510): “The Algi/zein (AP: MP) scaffold supports robust myotube alignment without external molds, indicating that morphological organization represents the primary demonstrable outcome under the present conditions.”

Comment 27

GraphPad Prism version. Methods say “GraphPad Prism 8” and “version 10.2.0”—resolve this discrepancy and specify OS, analysis modules, and multiple comparison tests.

Response 27:

The discrepancy in software version reporting has been corrected. All statistical analyses were performed using GraphPad Prism version 8.0.1.244 (64-bit, Windows). The Methods section has been updated accordingly, and the statistical tests and multiple-comparison procedures used for each analysis are now explicitly specified.

• Methods: Lines (286-293): “All quantitative data are presented as mean ± standard error (SE). Statistical analyses were performed using GraphPad Prism version 8.0.1.244 (64-bit, Windows). Data was analyzed using two-way ANOVA with repeated measures. When missing data points were present, mixed-effects models (REML) were applied. Post hoc comparisons were corrected using Holm–Šidák’s multiple-comparison tests, as appropriate. For each condition, n represents the number of individual hydrogel samples (scaffold replicates) analyzed per group (n = 4–8), as indicated in the figure legends. Adjusted p-values (q-values) < 0.05 were considered statistically significant.”

 

Comment 28

Terminology. Standardize “cultured meat” (not “culture meat” in keywords) and fix “Myomarker” → Myomaker in abbreviations.

Response 28:

Terminology has been standardized throughout the manuscript. “Cultured meat” is now used consistently (including in the keywords).

• Keywords: Line (27-28): “Keywords: Alginate; Adipocytes; C2C12; Cell powder; Cell culture; Cultured meat; Cell alignment; Zein”

Comment 29

Table 3 (GC–MS analysis). RT values like “RT 15365” seem to be typos

Response 29:

The retention time (RT) formatting errors in Table S1 have been corrected (“RT 15.365”), and the changes are highlighted in the revised table.

Comment 30

Identification is putative without m/z or MS/MS—label as MSI Level 2/3; move interpretive aroma notes to SI unless confirmed with standards.

Response 30:

The metabolomics identification confidence level has been revised for clarity. Because compound annotations were based on retention time matching without MS/MS fragmentation data or confirmation using authentic standards, all reported metabolites are now explicitly labeled as putative (MSI Level 3) in accordance with Metabolomics Standards Initiative guidelines.

• Methods: Lines (282-283): “Identifications were reported at MSI Level 3. Hierarchical clustering heatmaps were used for data visualization.”
• Results: Lines (530-544): “A hierarchical clustering heatmap was generated to compare metabolite profiles among beef, composite Algi/zein, and Algi/zein (AP:MP) scaffolds. The retention time (RT) values displayed distinct clustering patterns, indicating putative differences in metabolite composition across the groups (Figure 8). The Algi/zein (AP:MP) scaffold exhibited stronger signals at RT-1.296 and RT-1.076, approaching those of beef, suggesting the putative presence of metabolites associated with muscle tissue. In contrast, the Algi/zein scaffold showed moderate signals at RT-1.296 and RT-3.7042, with minimal activity at RT-1.076. Beef samples clustered separately, exhibiting a broader range of RT intensities, including high signals at RT-7.925 and RT-1.296, reflecting their native metabolic complexity. All peak assignments are putative (MSI Level 3) based on previously published retention times (Table S1), without confirmation by authentic standards. These results suggest that incorporation of AP and MP components alters the volatile metabolite profile of the scaffold toward patterns that partially overlap with beef, based on putative RT-based assignments.”
• Table S1

 

Comment 31

Distinguish process artifacts (ethanol from zein prep) from biological signals with proper controls.

Response 31:

Ethanol was used exclusively as a transient processing solvent during zein dissolution and was not intended to be a biological or structural component of the scaffold. Ethanol-related signals detected by GC–MS were observed across all lyophilized samples, including beef controls processed under identical conditions, indicating that these signals are most likely process-related artifacts rather than biologically derived metabolites. Accordingly, ethanol was not interpreted as a scaffold-derived or biologically meaningful feature. All metabolite annotations are reported as putative (MSI Level 3), and ethanol-related signals have been explicitly identified as process-associated in Table S1 to avoid overinterpretation.

• Table S1: “Detected in all samples. Ethanol was used only as a transient solvent during zein dissolution and was removed prior to cell culture and GC–MS analysis. Therefore, ethanol signals should not be interpreted as intrinsic scaffold-derived metabolites and likely reflect background or sample-processing-related traces [55].”

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript describes the development of alginate–zein hydrogels enriched with myotube and adipocyte cell powders and evaluates their structural, biological, and metabolomic properties as edible scaffolds to support C2C12 myoblast alignment and differentiation for cultured meat applications.

The topic is timely and relevant to the field of cultured meat and edible scaffold engineering. The integration of myotube and adipocyte powders into food-grade hydrogels is interesting; however, the overall level of innovation is moderate, as several scaffold-based approaches aiming at muscle alignment and biochemical mimicry have already been reported. The manuscript contains extensive experimental data but requires substantial revision to improve scientific rigor, reduce overinterpretation, and strengthen methodological clarity.

Major concerns are listed below.

• Lines 11–33 (Abstract): Reduce overstatement regarding “close mimicry” of real meat structure and metabolism.

• Lines 60–101 (Introduction): Clarify the specific scientific gap addressed by this study in relation to existing edible scaffolds for cultured meat.

• Lines 81–88: Moderate claims of novelty related to the use of myotube and adipocyte powders.

• Lines 112–127: Clarify ethical approval and regulatory considerations related to the use of murine cells for food-related applications.

• Lines 136–150: Clarify reproducibility and batch-to-batch variability of the cell powder preparation process.

• Lines 153–160: Clarify the rationale for the selected alginate and zein concentrations.

• Lines 162–169: Clarify the role of the food mold and its influence on cell alignment relative to scaffold composition.

• Lines 171–180: Clarify solvent use (DMSO) and its potential influence on cell responses.

• Lines 188–201: Clarify the experimental unit and number of independent replicates used for swelling and degradation analyses.

• Lines 202–209: Clarify how representative SEM images were selected and whether quantitative pore analysis was performed on multiple regions.

• Lines 210–221: Clarify whether cytotoxicity data were normalized to scaffold mass or surface area.

• Lines 224–237: Reduce descriptive interpretation of immunofluorescence images and avoid implying functional outcomes without quantitative confirmation.

• Lines 239–246: Clarify how residual nucleic acids from MP and AP were controlled in gene expression analysis.

• Lines 249–253: Moderate interpretations of FTIR data and avoid implying molecular interactions without supporting evidence.

• Lines 256–274: Clarify metabolite identification criteria and confidence levels for putative compound assignment.

• Lines 282–301: Moderate interpretations linking swelling and degradation behavior directly to improved nutrient diffusion.

• Lines 319–321: Rephrase conclusions implying superiority of Algi/zein over alginate without statistical confirmation.

• Lines 334–373: Reduce repetitive description of MP effects across viability, proliferation, and differentiation assays.

• Lines 389–391: Moderate conclusions regarding optimal AP:MP ratio and avoid generalization beyond the tested conditions.

• Lines 405–406: Avoid causal language when interpreting FTIR spectral differences.

• Lines 416–423: Reduce speculative interpretation of SEM images regarding ECM-like structure formation.

• Lines 429–434: Clarify the apparent inconsistency between qualitative myosin staining and non-significant gene expression results.

• Lines 435–449: Moderate claims regarding similarity of metabolite profiles to beef and avoid implying sensory equivalence.

• Lines 577–585 (Discussion): Reduce overgeneralization of applicability to structured cultured meat production.

Author Response

Comment 1
Lines 11–33 (Abstract): Reduce overstatement regarding “close mimicry” of real meat structure and
metabolism.
Response 1:
The abstract has been revised to reduce the overstatement and clarify the partial mimicry of our scaffold
to the beef tissue.
- Abstract : Lines (11-26): “Recent advances in cultured meat research emphasize the
development of edible scaffolds that promote myogenic differentiation. Nonetheless, many
materials provide only structural support and do not replicate native muscle or serve as
alternatives to muscle–adipocyte co-culture, highlighting the need for cytocompatible, tissuespecific
scaffolds. This study aimed to develop a composite alginate–zein (Algi/zein) hydrogel
enriched with myotube (MP) and adipocyte (AP) powders to provide a structural, bio-chemical,
and potential cultured-meat hydrogel. Algi/zein hydrogels enriched with myotube (MP) and
adipocyte (AP) powders were fabricated and evaluated for structural, cellular, and biochemical
properties using C2C12 myoblasts cultured in 2D and 3D environments. Metabolite profiling was
performed to evaluate the biochemical features. MP/AP incorporation generated ECM-like
microstructures and significantly enhanced myotube alignment in Algi/zein scaffolds compared
with MP/AP-free controls, increasing the proportion of axially aligned fibers by up to ~6-fold at a
1:1 AP: MP ratio. Organized myosin expression was observed, while metabolomic profiling
indicated partial biochemical similarity to beef. Incorporating MP and AP into Algi/zein hydrogels
enhanced myotube alignment and showed partial structural and biochemical similarity to native
muscle tissue.”

Comment 2
Lines 60–101 (Introduction): Clarify the specific scientific gap addressed by this study in relation to
existing edible scaffolds for cultured meat.
Response 2:
The Introduction has been updated to clearly define the scientific gap. Most edible scaffolds mainly
provide structural support. They often lack intrinsic myogenic and adipogenic biochemical cues in one
food-grade matrix. In addition, myotube alignment commonly requires external patterning methods, which
limit scalability. This study addresses these limitations by incorporating MP/AP into Algi/zein hydrogels to
provide intrinsic bioactivity and promote alignment.
- Introduction: Lines (91-110): “However, many edible scaffold systems still primarily provide
physical support and lack integrated lineage-specific bioactivity required for effective muscle
tissue mimicry [28, 37]. Together, these limitations underscore the need for strategies such as the
in-corporation of cell-derived components that enable scalable, bioactive, and food-grade scaffold
systems for fully biomimetic cultured meat [36]. Despite recent advances in edible scaffold
development, several critical limitations remain. First, most food-grade scaffolds primarily provide
mechanical support and lack lineage-specific biochemical bioactivity capable of simultaneously
delivering myogenic and adipogenic cues within a single matrix [44]. Second, the induction of
myotube alignment typically relies on external patterning strategies, molds, or mechanical
stimulation, which limits fabrication simplicity and scalability [45]. Third, many alignment-directing
scaffolds lack comprehensive food-grade biochemical vali-dation, restricting their translational
relevance to cultured-meat applications [44, 45]. Collectively, these limitations underscore the
need for edible scaffold systems that integrate intrinsic bioactivity, scalable self-alignment
capacity, and food-grade bio-chemical functionality. Accordingly, the primary objective of this
study was to evaluate whether the in-corporation of MP/AP into Algi/zein hydrogels enhances
myoblast alignment com-pared with MP/AP-free scaffolds. As a secondary objective, we further
assessed whether MP/AP-derived cues confer muscle-like biochemical characteristics.”

Comment 3
Lines 81–88: Moderate claims of novelty related to the use of myotube and adipocyte powders.
Response 3:
The Introduction has been revised to moderate novelty-related statements regarding the use of myotube
and adipocyte powders. In the updated version, MP/AP are presented as a proposed strategy rather than
a definitive novel breakthrough, and their expected role is framed as an objective and hypothesis. The
revised text is reflected in the following sentences included in the Introduction:
- Lines (93-96) :”Together, these limitations underscore the need for strategies such as the
incorporation of cell-derived components that enable scalable, bioactive, and food-grade scaffold
systems for fully biomimetic cultured meat [36].”
- Lines (107-114) : “Accordingly, the primary objective of this study was to evaluate whether the incorporation
of MP/AP into Algi/zein hydrogels enhances myoblast alignment com-pared with
MP/AP-free scaffolds. As a secondary objective, we further assessed whether MP/AP-derived
cues confer muscle-like biochemical characteristics. We hypothesized that incorporation of MP
and AP powders into Algi/zein hydro-gels would provide intrinsic biochemical and topographical
cues sufficient to enhance myotube alignment without the need for external patterning, while also
introducing muscle-like biochemical features.”

Comment 4
Lines 112–127: Clarify ethical approval and regulatory considerations related to the use of murine
cells for food-related applications.
Response 4:
The Methods and Discussion sections have been updated to clearly state the ethical approval
and regulatory considerations for the use of murine-derived cells. All mouse procedures were
performed in accordance with institutional guidelines and were approved by the Institutional
Animal Care and Use Committee (IACUC). The corresponding IACUC approval statement has
also been included in the manuscript (Institutional Animal Care and Use Committee Statement
section). In addition, we clarified that murine preadipocytes were used solely as an in vitro
research model for scaffold evaluation and not for direct food production or regulatory
submission.
- Methods: Lines (149-151): “Murine preadipocytes were used only as an in vitro research model to
evaluate scaffold bioactivity and adipogenic differentiation, and this study is not intended for direct
food production or regulatory submission.”
- Discussion : Lines (680-685): “These limitations highlight the need for further validation using
livestock-derived cells, functional assays, and comprehensive food-grade evaluations. Finally, the
absence of mechanical stimulation, long-term maturation analysis, and in vivo validation, together
with the use of murine rather than primary bovine myoblasts, may limit the direct translational
scalability of this platform to industrial cultured meat production.”
- Institutional Review Board Statement: Lines (716-718): “All animal procedures were approved by
the Institutional Animal Care and Use Committee of Chungbuk National University (Approval No.
CBNUA-25-0053-02).”

Comment 5
Lines 136–150: Clarify reproducibility and batch-to-batch variability of the cell powder preparation
process.
Response 5:
The Methods section has been updated to clarify the reproducibility of the cell powder
preparation. MP and AP were produced in two independent batches using identical culture and
lyophilization conditions (10 dishes per batch), yielding consistent dry powder mass (~1.25–1.30
g). In addition, FTIR was performed on each powder and on powder-supplemented scaffolds to
confirm comparable chemical features between batches.
- Methods: Lines (168-170): “Cell powders were prepared in two independent batches using the
same protocol. In each batch, MP and AP were produced separately from 10 dishes (150 × 25
mm) per cell type. The final dry powder yield was consistent between batches (~1.25–1.30 g per
batch).”

Comment 6
Lines 153–160: Clarify the rationale for the selected alginate and zein concentrations.
Response 6:
The Methods section has been updated to clarify that alginate and zein concentrations were
selected based on the reference formulation [46] and optimized within a stable range to ensure
homogeneous hydrogel formation. Alginate provided the main gel-forming network, while zein
was included at a level sufficient to improve matrix reinforcement and protein-based cues without
compromising uniformity or handling.
- Methods : Lines (174-181): “The alginate and zein concentrations were selected based on the
reported formulation [46]. This composition ensured stable gelation and homogeneous mixing for
cell culture. Briefly, 0.4 g of zein (Sigma Aldrich) was dissolved in 20 ml of 75% ethanol, then 10
ml of the resolved zein was added to 30 ml of 1% Algi (Sigma Aldrich) dis-solved in 50°C distilled
water (DW). Ethanol was used only as a transient solvent and was removed before analysis. The
homogenate of Algi/zein hydrogel was stirred using a magnetic bar at 50°C. Then, 40 ml of the
Algi solution was added to the homogenate, reaching a concentration of 0.28%. The Algi/zein
hydrogel was preserved in the refrigerator for 7 days.”

Comment 7
Lines 162–169: Clarify the role of the food mold and its influence on cell alignment relative to
scaffold composition.
Response 7:
All 3D experiments reported in the revised manuscript were conducted without external molds or
patterning devices. The previously included food-grade mold experiment (Submitted manuscript)
was part of an early optimization step and has been fully removed to avoid confusion. The
Introduction, Discussion, and Conclusions have been revised to ensure that all statements
regarding spontaneous alignment are based exclusively on mold-free 3D experiments and on the
direct comparison against MP/AP-free Algi/zein scaffolds.
- Introduction: Lines (111-114): “We hypothesized that incorporation of MP and AP powders into
Algi/zein hydrogels would provide intrinsic biochemical and topographical cues sufficient to
enhance myotube alignment without the need for external patterning, while also introducing
muscle-like biochemical features.”
- Discussion: Lines (672-673):“Muscle differentiation was inferred solely from morphological
alignment and myosin immunostaining.”
- Conclusion: Lines (687-690): “This study demonstrates that MP/AP-enriched Algi/zein hydrogels
enable robust intrinsic myotube alignment without external molds while maintaining
cytocompatible growth of C2C12 myoblasts, representing the primary functional outcome of the
scaffold system. “

Comment 8
Lines 171–180: Clarify solvent use (DMSO) and its potential influence on cell responses.
Response 8:
AP was dissolved in DMSO because it did not dissolve directly in culture medium, resulting in a final
DMSO concentration of 0.1% (v/v). This concentration is commonly used in cell culture and is generally
considered non-cytotoxic (Reference 68). In the present study, Live/Dead staining, the CCK-8 assay, and
PAX7 immunofluorescence (Figure 5a–b) showed no adverse effects under the tested conditions,
indicating that 0.1% DMSO did not measurably alter cell viability or behavior in this system. This
clarification has been added to the revised manuscript.
- Methods: Lines (197-199): “While AP was dissolved in 0.1% Dimethyl sulfoxide (DMSO) (Sigma
Aldrich) before preparing AP: MP ratios. The final DMSO concentration in all culture conditions did
not exceed 0.1% (v/v).”
- Discussion: Lines (598-601): “Although AP was dissolved in 0.1% DMSO for preparation purposes,
this low concentration is widely tolerated in in vitro systems [68] and did not interfere with
cytocompatibility or myogenic differentiation under the present experimental conditions.”
- Reference (68) Figure 1A-C

Comment 9
Lines 188–201: Clarify the experimental unit and number of independent replicates used for swelling and
degradation analyses.
Response 9:
The Methods section has been updated to clarify the experimental unit and replicates. The experimental
unit was one scaffold. Swelling and degradation were performed using n = 8 independent scaffolds per
group per time point, with separate scaffold sets used for each assay.
- Methods: Lines (225-227): “The experimental unit was one individual hydrogel scaffold. Swelling
and degradation were measured using n = 8 independent scaffolds per group per time point
(destructive sampling).”

Comment 10
Lines 202–209: Clarify how representative SEM images were selected and whether quantitative pore
analysis was performed on multiple regions.
Response 10:
The Methods section has been updated to clarify SEM image selection and pore quantification.
Quantitative pore analysis was performed using three non-overlapping SEM fields of view per scaffold (n
= 3). Representative images were selected from these analyzed fields and reflect typical scaffold
morphology.
- Methods : Lines (234-237): “For each scaffold, pore quantification was performed using three
SEM images from three different, non-overlapping fields of view (n = 3 fields per scaffold).
Representative images were selected from these fields based on typical morphology.”

Comment 11
Lines 210–221: Clarify whether cytotoxicity data were normalized to scaffold mass or surface area.
Response 11:
The Methods section has been updated to clarify normalization. Cytotoxicity was evaluated under
identical culture conditions (same seeding density and culture volume) across all MP/AP
concentrations; therefore, CCK-8 absorbance values were not normalized to surface area.
- Methods: Lines (250-252): “Absorbance values were not normalized to surface area because all
groups were tested under identical culture conditions using the same seeding density and culture
volume.”
Scaffold cytotoxicity data were also generated in the preliminary mold-based optimization study.
However, the dataset has been removed from the revised manuscript to avoid confusion. Cytotoxicity
(CCK-8) results were not normalized to scaffold mass or surface area because all scaffolds were
prepared with the same dimensions and were tested under the same conditions.

Comment 12
Lines 224–237: Reduce descriptive interpretation of immunofluorescence images and avoid implying
functional outcomes without quantitative confirmation.
Response 12:
The Results section has been revised to reduce descriptive interpretation of immunofluorescence images
and to avoid implying functional outcomes unless supported by quantitative analysis.
- Lines (354-360): “Immunostaining of the proliferation marker PAX-7 showed that C2C12 cells
were proliferative within both Algi/zein and Algi hydrogels, observed over the first five days
(Figure 2a). During the proliferation phase, immunofluorescence staining for the early myogenic
marker Myo-d revealed the initiation of C2C12 differentiation on days 3 and 5 (Figure 2b). After
differentiation induction, the Immunostaining of the late differentiation marker myosin showed
multinucleated myotubes in Algi/zein hydrogel preferentially than in Algi (Figure 2c). Those
findings suggest that Algi/zein hydrogel supported C2C12 proliferation and differentiation.”
- Lines (412-420): “Myogenic differentiation was assessed by desmin immunostaining. MP
supplementation up to 0.1 μg/μL increased myotube formation and improved myotube
organization compared with the control group, which showed scarce and disorganized desminpositive
fibers (Figure 4d; Figure S2). The 0.05 and 0.1 μg/μL MP groups exhibited denser and
more aligned myotubes, indicating enhanced structural maturation. Myotube orientation was
further quantified (Figure 4e), showing that 0.1 μg/μL MP resulted in a greater proportion of
parallel-aligned myotubes. Collectively, these findings indicate that MP at 0.1 μg/μL supports
cytocompatibility and promotes organized desmin-positive myotube formation.”
- Lines (441-452): “Immunostaining for desmin was used to visualize cytoskeletal organization and
myogenic differentiation. MP: AP -treated cells (1:1, 2:1, and 4:1) exhibited more uniform desmin
organization compared with control (1:0), whereas a higher MP: AP ratio (6:1) was associated
with disrupted desmin integrity (Figure 5d). Myotube alignment (des-min⁺ myotubes) was
analyzed from −90° to 90°, and the fusion index at 0° was used as the primary orientation
endpoint. All MP/AP-enriched media showed a general alignment shift toward ~30° angle. Among
these, the 1:1 AP: MP formulation exhibited the greatest enrichment of axially aligned fibers (0° ±
10°). Specifically, the 1:0, 1:1, 2:1, 4:1, and 6:1 AP: MP groups showed 125%, 617%, 136%,
512%, and 169% , respectively (Figure 5e). Overall, under the tested conditions, the 1:1 AP: MP
ratio showed the highest enrichment of axially aligned myotubes while maintaining cell viability,
suggesting it as a suitable working ratio for subsequent experiments.”
- Lines (512-519): “Immunofluorescence staining of myosin demonstrated differential myogenic
activity between Algi/zein and Algi/zein (AP: MP) (Figure 7c). In the Algi/zein (AP: MP) scaffold,
myosin expression displayed highly aligned myotube formation with abundant nuclei, indicative of
mature myotube structure. Conversely, the Algi/zein (free powder) scaffold showed weak and
disorganized myosin expression, with no evidence of aligned myotube structures. The Algi/zein
(AP: MP) scaffold supports robust myotube alignment without external molds, indicating that
morphological organization represents the primary demonstrable outcome under the present
conditions.”

Comment 13
Lines 239–246: Clarify how residual nucleic acids from MP and AP were controlled in gene
expression analysis.
Response 13:
Residual nucleic acids from MP/AP were controlled by applying an optimized DNase I
pretreatment before RNA extraction. However, qPCR experiments were repeated (at the revision
time) the analysis did not yield statistically significant gene expression differences. Accordingly, to
avoid overinterpretation, the qPCR results were removed from the manuscript in their entirety,
and this limitation has been stated in the revised Discussion.
- Discussion: Lines (Lines 673–676): “Although trends were observed, the lack of qPCR analysis
under the present experimental conditions indicates the need for larger sample sizes and additional
markers (e.g., TnT, MHC isoforms) in future studies.”

Comment 14
Lines 249–253: Moderate interpretations of FTIR data and avoid implying molecular interactions
without supporting evidence.
Response 14:
The FTIR Results and Discussion have been revised to moderate interpretations. Peak
assignments are presented as functional-group evidence supported by references (Table 1), and
any mechanistic interaction statements were rephrased as putative.
- Results: Lines (468-479): “FTIR spectroscopy was employed to characterize the chemical features
and compositional differences of Algi/zein and Algi/zein (AP:MP) hydrogels (Figure 6). All spectra
showed broad absorption bands around 3270–3300 cm⁻¹, indicating O–H and N–H stretching
vibrations associated with proteins and water. The MP spectrum displayed prominent peaks at
~1650 cm⁻¹ (amide I, C=O stretching) and ~1540 cm⁻¹ (amide II, N–H bending), consistent with
protein presence [49]. In contrast, the AP spectrum showed weaker amide bands but more
pronounced absorptions in the region of 1200–2000 cm⁻¹, likely associated with C–O stretching of
carbohydrates and lipid-related groups. The control sample (Algi/zein) exhibited lower overall
intensity and fewer distinct absorption peaks. These spectral features reflect the unique molecular
compositions of the Algi/zein (AP: MP) hydrogel. MP is protein-dominant, whereas AP shows lipid
and carbohydrate signatures (Table 1).”
- Discussion: Lines (568-574): “This study demonstrates that incorporating MP and AP powders into
Algi/zein hydrogels can generate food-relevant scaffolds that partially mimic muscle-related
structural and biochemical features in vitro. The composite scaffolds supported C2C12 alignment
and myotube formation, and RT-based volatile profiling showed partial overlap with beef patterns.
These findings suggest MP/AP supplementation as a potential strategy to enhance scaffold
bioactivity; however, further validation under scalable cultured-meat processing conditions is
required. “
- Discussion: Lines (678-680): “While metabolomic profiling suggested partial biochemical similarity
to native beef tissue, compound identification remained putative, limiting chemical interpretation.”

Comment 15
Lines 256–274: Clarify metabolite identification criteria and confidence levels for putative
compound assignment.
Response 15:
Metabolite identities were assigned based on previously published retention times (RT) and mass
spectral data (references provided in Table S1), rather than by comparison to spectral libraries (e.g.,
NIST/Wiley) or authentic standards. Therefore, all metabolite identifications are reported as putative
and correspond to MSI Level 3. The Methods and Results sections have been revised to explicitly state
the identification confidence level and to avoid overinterpretation of these assignments.
- Methods: Lines (297-298): “Identifications were reported at MSI Level 3. Hierarchical clustering
heatmaps were used for data visualization.”
- Results: Lines (549-553): “All peak assignments are putative (MSI Level 3) based on previously
published retention times (Table S1), without confirmation by authentic standards. These results
suggest that incorporation of AP and MP components alters the volatile metabolite profile of the
scaffold toward patterns that partially overlap with beef, based on putative RT-based
assignments.”
- Table S1 note: ”All metabolite identifications are putative (MSI Level 3) and were assigned based
on previously reported retention times. No authentic standards, MS/MS fragmentation, or library
match scores were obtained.”

Comment 16
Lines 282–301: Moderate interpretations linking swelling and degradation behavior directly to improved
nutrient diffusion.
Response 16:
The Results section has been revised to moderate interpretation and to avoid directly linking swelling
and degradation behavior to improved nutrient diffusion. The revised text now reports the measured
swelling and biodegradation outcomes and attributes the reduced swelling to zein hydrophobicity,
without implying functional transport effects that were not directly quantified.
- Results: Lines (311-321): “Over 10 days, Algi/zein hydrogel exhibited significantly lower swelling
(1265.41 ± 191.67) compared to Algi hydrogel (2947.92 ± 252.95) (Figure. 1a), indicating reduced
water uptake following incorporation of zein. This reduced swelling is consistent with the
hydrophobic nature of zein and suggests that zein incorporation modulates the hydration behavior
of the Algi-based scaffold. Biodegradation of Algi and Algi/zein hydrogels was evaluated over the
same 10-day period. Although Algi/zein hydrogels showed a higher mean biodegradation
percentage (21.38 ± 2.16%) compared with Algi (14.21 ± 1.23%), no statistically signifi-cant
differences were observed at any time point (Figure 1b). These results indicate that incorporation
of zein alters swelling behavior without substantially affecting the overall degradation profile of the
Algi-based hydrogel.”

Comment 17
Lines 319–321: Rephrase conclusions implying superiority of Algi/zein over alginate without statistical
confirmation.
Response 17:
The Results interpretation was revised to avoid implying superiority of Algi/zein over Algi without
statistical support. Statements suggesting preferential myotube formation in Algi/zein were rephrased
to report qualitative observations only, and the text now indicates that both Algi/zein and alginate
hydrogels supported C2C12 proliferation and myogenic differentiation.
- Results: Lines (354-360): “Immunostaining of the proliferation marker PAX-7 confirmed the
presence of proliferative C2C12 cells in both Algi/zein and Algi hydrogels during the first 5 days
(Figure 2a). During the proliferation phase, MyoD staining indicated initiation of myogenic
differentiation on days 3 and 5 (Figure 2b). After differentiation induction, myosin staining showed
multinucleated myotube formation in both hydrogel groups (Figure 2c). Overall, these results
indicate that both Algi/zein and Algi hydrogels supported C2C12 proliferation and myogenic
differentiation.”

Comment 18
Lines 334–373: Reduce repetitive description of MP effects across viability, proliferation, and
differentiation assays.
Respond 18:
The Results section describing MP effects has been revised to reduce repetition across viability,
proliferation, and differentiation assays. The text was condensed to report cytocompatibility findings
(Live/Dead and CCK-8) together. Followed by differentiation outcomes (desmin staining and orientation
analysis), while maintaining all quantitative values and figure references.
- Results: Lines (400-420): “The effects of MP on C2C12 cell viability and proliferation were
evaluated in 2D culture using Live/Dead staining and CCK-8 assays. Live/Dead staining
demonstrated high cell viability across all MP concentrations tested (0.01, 0.05, and 0.1 μg/μL),
indicating cytocompatibility of the MP-enriched environment (Figure 4a). The immuno-fluorescent
staining of PAX-7 indicates the maintenance of proliferated C2C12 cells after 3 days (Figure 4b).
Consistently, CCK-8 analysis showed no cytotoxic effects at any concentration, with a nonsignificant
increase in viability observed at 0.1 μg/μL MP (114.16 ± 3.27%) compared with 0.05
μg/μL (100.42 ± 3.99%) and 0.01 μg/μL (106.94 ± 4.97%) (Figure 4c). In contrast, higher MP
concentrations (>0.1 μg/μL; 0.2, 0.3, and 0.4 μg/μL) induced marked cytotoxicity and significant
cell death over 5 days (Figure S1a and b), defining 0.01–0.1 μg/μL as the working concentration
range. Myogenic differentiation was assessed by desmin immunostaining. MP supplementation
up to 0.1 μg/μL increased myotube formation and improved myotube organization compared with
the control group, which showed scarce and disorganized desmin-positive fibers (Figure 4d;
Figure S2). The 0.05 and 0.1 μg/μL MP groups exhibited denser and more aligned myotubes,
indicating enhanced structural maturation. Myotube orientation was further quantified (Figure 4e),
showing that 0.1 μg/μL MP resulted in a greater proportion of parallel-aligned myotubes.
Collectively, these findings indicate that MP at 0.1 μg/μL supports cytocompatibility and promotes
organized desmin-positive myotube formation.”

Comment 19
Lines 389–391: Moderate conclusions regarding optimal AP:MP ratio and avoid generalization
beyond the tested conditions.
Response 19:
The Results section was revised to moderate statements regarding the AP: MP ratio. The revised
text now describes the 1:1 ratio as a suitable working condition under the tested experimental
settings, rather than claiming an “optimal” ratio or generalizing beyond the evaluated
concentrations and time points.
- Results: Lines (441-452): “Immunostaining for desmin was used to visualize cytoskeletal
organization and myogenic differentiation. MP: AP -treated cells (1:1, 2:1, and 4:1) exhibited more
uniform desmin organization compared with control (1:0), whereas a higher MP: AP ratio (6:1)
was associated with disrupted desmin integrity (Figure 5d). Myotube alignment (des-min⁺
myotubes) was analyzed from −90° to 90°, and the fusion index at 0° was used as the primary
orientation endpoint. All MP/AP-enriched media showed a general alignment shift toward ~30°
angle. Among these, the 1:1 AP: MP formulation exhibited the greatest enrichment of axially
aligned fibers (0° ± 10°). Specifically, the 1:0, 1:1, 2:1, 4:1, and 6:1 AP: MP groups showed 125%,
617%, 136%, 512%, and 169% , respectively (Figure 5e). Overall, under the tested conditions,
the 1:1 AP: MP ratio showed the highest enrichment of axially aligned myotubes while maintaining
cell viability, suggesting it as a suitable working ratio for subsequent experiments.”

Comment 20
Lines 405–406: Avoid causal language when interpreting FTIR spectral differences.
Respond 20:
The FTIR Results section has been revised to avoid causal language when interpreting spectral
differences. Peak assignments are now described using neutral terms.
- Results: Lines (468-479): “FTIR spectroscopy was employed to characterize the chemical features
and compositional differences of Algi/zein and Algi/zein (AP:MP) hydrogels (Figure 6). All spectra
showed broad absorption bands around 3270–3300 cm⁻¹, indicating O–H and N–H stretching
vibrations associated with proteins and water. The MP spectrum displayed prominent peaks at
~1650 cm⁻¹ (amide I, C=O stretching) and ~1540 cm⁻¹ (amide II, N–H bending), consistent with
protein presence [49]. In contrast, the AP spectrum showed weaker amide bands but more
pronounced absorptions in the region of 1200–2000 cm⁻¹, likely associated with C–O stretching of
carbohydrates and lipid-related groups. The control sample (Algi/zein) exhibited lower overall
intensity and fewer dis-tinct absorption peaks. These spectral features reflect the unique molecular
compositions of the Algi/zein (AP: MP) hydrogel. MP is protein-dominant, whereas AP shows lipid
and carbohydrate signatures (Table 1).”

Comment 21
Lines 416–423: Reduce speculative interpretation of SEM images regarding ECM-like structure
formation.
Respond 21:
The SEM Results section has been revised to reduce speculative interpretation. The revised text
now reports the morphological observations in neutral terms and describes ECM-like features as
aligned fibrous structures without implying molecular identity or functional outcomes.
- Results: Lines (499-511) : “After 10 days of C2C12 differentiation within the hydrogel scaffolds,
morphological characterization showed differences between Algi/zein and Algi/zein (AP:MP)
scaffolds. The Algi/zein (AP:MP) scaffold exhibited a smoother and more striated surface compared
with the relatively diffuse Algi/zein hydrogel (Figure 7a), indicating in-creased structural
compactness after AP/MP incorporation. SEM imaging supported these observations (Figure 7b).
The plain (cell-free) Algi/zein scaffold displayed an irregular porous network with a heterogeneous
surface texture. After C2C12 culture, additional fibrous features were observed on the scaffold
surface (Figure S3a). In contrast, the plain Algi/zein (AP:MP) scaffold showed a more aligned
fibrous architecture. Following C2C12 culture in Algi/zein (AP:MP), these fibrous structures were
more pronounced, and aligned bundle-like features were observed (Figure 7b). Similar
morphologies were detected across different fields of view (Figure S3b). Overall, these results
indicate that AP/MP incorporation was associated with increased surface alignment and structural
organization of the scaffold after culture.”

Comment 22
Lines 429–434: Clarify the apparent inconsistency between qualitative myosin staining and nonsignificant
gene expression results.
Respond 22:
To avoid overinterpretation, the gene expression dataset was removed from the revised manuscript
because repeated analysis did not confirm statistically significant differences. The Results and
Discussion now rely on the qualitative myosin immunostaining findings only, and no conclusions
are drawn regarding transcriptional upregulation. This limitation has been explicitly acknowledged
in the Discussion section.
- Discussion: Lines (672-676): “Muscle differentiation was inferred solely from morphological
alignment and myosin immunostaining. Although trends were observed, the lack of qPCR analysis
under the present experimental conditions indicates the need for larger sample sizes and additional
markers (e.g., TnT, MHC isoforms) in future studies. “

Comment 23
Lines 435–449: Moderate claims regarding similarity of metabolite profiles to beef and avoid
implying sensory equivalence.
Respond 23:
The interpretation was revised to moderate claims regarding similarity to beef and to avoid implying
sensory equivalence. We now describe only partial overlaps in RT-based volatile patterns and
explicitly state that all metabolite annotations are putative (MSI Level 3) without confirmation using
authentic standards.
- Results: Lines (539-553): “A hierarchical clustering heatmap was generated to compare
metabolite profiles among beef, composite Algi/zein, and Algi/zein (AP:MP) scaffolds. The
retention time (RT) values displayed distinct clustering patterns, indicating putative differences in
metabolite composition across the groups (Figure 8). The Algi/zein (AP:MP) scaffold exhibited
stronger signals at RT-1.296 and RT-1.076, approaching those of beef, suggesting the putative
presence of metabolites associated with muscle tissue. In contrast, the Algi/zein scaffold showed
moderate signals at RT-1.296 and RT-3.7042, with minimal activity at RT-1.076. Beef samples
clustered separately, exhibiting a broader range of RT intensities, including high signals at RT-
7.925 and RT-1.296, reflecting their native metabolic complexity. All peak assignments are
putative (MSI Level 3) based on previously published retention times (Table S1), without
confirmation by authentic standards. These results suggest that incorporation of AP and MP
components alters the volatile metabolite profile of the scaffold toward patterns that partially
overlap with beef, based on putative RT-based assignments.”

Comment 24
Lines 577–585 (Discussion): Reduce overgeneralization of applicability to structured cultured
meat production.
Respond 24:
The Discussion has been revised to reduce overgeneralization regarding applicability to
structured cultured meat production. The revised text now limits conclusions to the tested in vitro
conditions and frames cultured-meat relevance as potential, rather than demonstrated scalability
or production readiness.
- Discussion: Lines: (568-574): “This study demonstrates that incorporating MP and AP powders
into Algi/zein hydrogels can generate food-relevant scaffolds that partially mimic muscle-related
structural and biochemical features in vitro. The composite scaffolds supported C2C12 alignment
and myotube formation, and RT-based volatile profiling showed partial overlap with beef patterns.
These findings suggest MP/AP supplementation as a potential strategy to enhance scaffold
bioactivity; however, further validation under scalable cultured-meat processing conditions is
required.”

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors responded to my suggestions and they have addressed all the comments appropriately. In my opinion, the manuscript is now ready for acceptance.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have adequately addressed all major concerns raised during peer review by substantially moderating overinterpretation, improving methodological clarity, and clearly redefining the scope of the study. The revised manuscript now presents a scientifically sound proof-of-concept and is suitable for publication in its current form. Thanks!