Enhancing Glucuronic Acid and Bacterial Cellulose Yield in Kombucha via Valorization of Male Jelly Fig (Ficus pumila L. var. awkeotsang)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article addresses the timely and interesting topic of valuing an underappreciated plant material in kombucha production and assessing the co-formed bacterial cellulose. In its current form, the manuscript requires major revision before possible publication. Comments are provided below.
General comments:
1. The Materials and Methods chapter is one of the weakest sections of the manuscript. The procedure description is not detailed enough to fully replicate the experiment in another laboratory. It lacks, among other things, the full composition of the activation media, inoculum standardization, details of tea preparation, HPLC parameters, and other information.
2. The methodological section contains a significant formal error: the formula for the swelling ratio appears incorrect because it does not use the Ws variable, even though it was previously defined as swollen mass.
3. The discussion is sufficiently long, but it contains too many speculative statements. In several places, the authors suggest mechanisms they did not investigate, such as those related to caffeine utilization by microorganisms, vitamin C stabilization, or the biological significance of succinate. The discussion should be more grounded in the data obtained in this work.

Specific comments:
• Lines 33–35: "This study evaluated male fig powder (0–2%, w/v) as a substrate for developing male fig–altered kombucha (FK) using a defined (symbiotic culture of bacteria and yeast) SCOBY inoculum..." This study used a system composed of two isolated species, not a classic, complex SCOBY. Please consider changing it to: "This study evaluated male fig powder (0–2%, w/v) as a substrate for producing male fig–altered kombucha (FK) using a defined co-culture of Komagataeibacter xylinus and Saccharomyces cerevisiae."
• Lines 42–43: “Antioxidant capacity (DPPH) and total phenolics (TPC) increased numerically…” — this sentence highlights an effect that was not statistically significant. Please change this to: “Numerical increases in DPPH and TPC were observed; however, no significant differences were detected among treatments.”
• Lines 57–59: “kombucha has attracted attention for multiple bioactivities… please change this to “kombucha has been investigated for several reported bioactivities…”
• Lines 64–65: “organic acids formed by lactic acid bacteria and AAB…” — a system involving LAB was not investigated in this study.
• Lines 85–87: “scientific information on their composition and functional potential remains limited…” — Please add specific literature data regarding the chemical composition of Male syconia, e.g., pectin, phenol, or sugar content.
• Lines 127–135: the HPLC description is too general. The instrument model, detector type, injection volume, analyte retention time, etc., are missing.
• Lines 136–144: The description of the DPPH assay requires clarification. Please provide the microplate reader model, the number of technical replicates, and the method for preparing the Trolox curve.
• Lines 145–152: The TPC unit "mg GAE/g" is unclear for liquid samples. Please clarify the mass to which the result refers, or consider reporting results in mg GAE/L.
• Lines 158–159: "reported per batch (or normalized to culture volume, where applicable)"—the wording is ambiguous. Please use one consistent method for reporting yield and use it consistently throughout the text.
• Lines 163–166: The formula for the swelling ratio appears incorrect because it uses the same variables as water content. Please correct the equation according to the previously defined Ws variable. This is a significant methodological error.
• Lines 198–209: The description of metabolite changes in Results is correct, but is primarily narrative in nature. Please emphasize that the observed trends are not always linear with the dose of Male Fig Powder.
• Lines 223–228: "Numerically, all FK groups showed higher mean DPPH values..." — again, a statistically insignificant effect is highlighted...
• Lines 279–286: The caption for Figure 2 includes "PK fermentations" and "PK fermentation," which is most likely an editorial error. Please correct this to "FK fermentations" or another consistent name used in the manuscript. • Lines 320–321: “Succinate is additionally recognized as a microbiota-associated metabolite involved in host metabolic signaling…” — This fragment is a digression and does not contribute much to the interpretation of the results presented in the paper.
• Lines 322–325: “Which may reflect matrix-dependent partitioning or differences in microbial utilization…” — This is a speculative hypothesis, not supported by any additional data. Please clearly mark this as a possible explanation, not an interpretation derived directly from the experiment.
• Lines 351–370: The discussion regarding KBC is interesting, but requires better comparison with previous studies on the effects of different fermentation matrices on the yield and properties of BC. The current literature is too general. The literature has already described both the effect of various herbal infusions on the production of BC films and the use of decomposed products.

Author Response

General comments:
1. The Materials and Methods chapter is one of the weakest sections of the manuscript. The procedure description is not detailed enough to fully replicate the experiment in another laboratory. It lacks, among other things, the full composition of the activation media, inoculum standardization, details of tea preparation, HPLC parameters, and other information.
[Response]: We thank the reviewer for this important comment. We have substantially expanded the Materials and Methods section to provide sufficient detail for reproducibility. Specifically, we added the full formulations of the activation media, inoculum preparation and standardization procedures, detailed tea preparation and sterilization steps, and comprehensive analytical parameters for HPLC, antioxidant assays, and physicochemical characterization.

 

2. The methodological section contains a significant formal error: the formula for the swelling ratio appears incorrect because it does not use the W_s variable, even though it was previously defined as swollen mass.

[Response]: We thank the reviewer for pointing out this important inconsistency. We agree and have corrected the swelling-ratio calculation to explicitly use the swollen mass (W_s) as defined in the Methods. In the revised manuscript, we also clarified the experimental workflow by adding a re-drying step after the swelling measurement: following the recording of W_s, samples were freeze-dried again to constant mass and reweighed to obtain the re-dried mass (W_d,re). This additional step was included to document mass stability after rehydration; however, the swelling ratio itself is calculated using W_s and the initial dry mass (W_d) according to the corrected equation.

 

3. The discussion is sufficiently long, but it contains too many speculative statements. In several places, the authors suggest mechanisms they did not investigate, such as those related to caffeine utilization by microorganisms, vitamin C stabilization, or the biological significance of succinate. The discussion should be more grounded in the data obtained in this work.

[Response]: We appreciate this suggestion and agree. We have revised the Discussion to reduce speculative statements and to focus on interpretations supported by the data. Statements regarding microbial caffeine utilization, vitamin C stabilization mechanisms, and the biological significance of succinate were either removed or rephrased as hypotheses with appropriate caveats and literature support.

 

Specific comments:
• Lines 33–35: "This study evaluated male fig powder (0–2%, w/v) as a substrate for developing male fig–altered kombucha (FK) using a defined (symbiotic culture of bacteria and yeast) SCOBY inoculum..." This study used a system composed of two isolated species, not a classic, complex SCOBY. Please consider changing it to: "This study evaluated male fig powder (0–2%, w/v) as a substrate for producing male fig–altered kombucha (FK) using a defined co-culture of Komagataeibacter xylinus and Saccharomyces cerevisiae."
[Response]: Lines 34–36: We thank the reviewer for this helpful comment. We agree and have revised the wording throughout the manuscript to accurately describe the inoculum as a defined co-culture rather than a complex SCOBY.

 

• Lines 42–43: “Antioxidant capacity (DPPH) and total phenolics (TPC) increased numerically…” — this sentence highlights an effect that was not statistically significant. Please change this to: “Numerical increases in DPPH and TPC were observed; however, no significant differences were detected among treatments.”
[Response]: Lines 42–43: We appreciate this suggestion. We agree and have revised the sentence to reflect the statistical outcome.

 

•Lines 57–59: “kombucha has attracted attention for multiple bioactivities… please change this to “kombucha has been investigated for several reported bioactivities…”
[Response]:Lines 56–57: Thank you for this valuable recommendation. We agree and revised the wording as suggested.

 

•Lines 64–65: “organic acids formed by lactic acid bacteria and AAB…” — a system involving LAB was not investigated in this study.
[Response]: Line 63: We are grateful to the reviewer for pointing this out. We agree and removed references to lactic acid bacteria from the mechanistic description, since LAB were not included in the defined co-culture used here.

 

•Lines 85–87: “scientific information on their composition and functional potential remains limited…” — Please add specific literature data regarding the chemical composition of Male syconia, e.g., pectin, phenol, or sugar content.
[Response]: Lines 85–88: Thank you for highlighting this important point. We added literature-based composition information available for Ficus pumila var. awkeotsang syconium/achenes, including pectin/methoxy-related parameters, to better contextualize the substrate. We also clarified that detailed chemical composition data specifically for male syconia remain limited and constitute a motivation for this work.

 

•Lines 127–135: the HPLC description is too general. The instrument model, detector type, injection volume, analyte retention time, etc., are missing.
[Response]: Lines 131–141: We appreciate this thoughtful comment, which helped us improve the clarity of the manuscript. We agree and expanded the HPLC method description to include instrument model, detector type, and injection volume for each analyte.

 

•Lines 136–144: The description of the DPPH assay requires clarification. Please provide the microplate reader model, the number of technical replicates, and the method for preparing the Trolox curve.
[Response]: Lines 144–153: We thank the reviewer for this helpful suggestion. We agree and have expanded the DPPH method by specifying the microplate reader model, the number of technical replicates, and the preparation range of the Trolox calibration curve used for TEAC quantification.

 

•Lines 145–152: The TPC unit "mg GAE/g" is unclear for liquid samples. Please clarify the mass to which the result refers, or consider reporting results in mg GAE/L.
[Response]: Line 161: We thank the reviewer for this important comment. We agree that reporting TPC as mg GAE/g is ambiguous for liquid samples. In the revised manuscript, TPC was quantified against a gallic acid calibration curve and is now reported as mg gallic acid equivalents per milliliter of kombucha (mg GAE/mL). For transparency, we also clarified the basis of the conversion: because the original values were expressed on a mass basis for liquid samples, they were converted assuming a sample density of 1.00 g/mL (i.e., mg GAE/g ≈ mg GAE/mL), and then converted to mg GAE/mL. Accordingly, the Methods section and Table 2 have been updated.

 

•Lines 158–159: "reported per batch (or normalized to culture volume, where applicable)"—the wording is ambiguous. Please use one consistent method for reporting yield and use it consistently throughout the text.
[Response]: Lines 167–168: We thank the reviewer for this helpful comment. We agree and have revised the manuscript to use a single, consistent definition of BC yield throughout. BC yield is now reported exclusively as freeze-dried mass normalized to fermentation volume (g/L).

 

•Lines 163–166: The formula for the swelling ratio appears incorrect because it uses the same variables as water content. Please correct the equation according to the previously defined W_s variable. This is a significant methodological error.
[Response]: Lines 168–176: We thank the reviewer for carefully identifying this important error. We agree and have corrected the swelling ratio equation to explicitly use the swollen mass (W_s) as previously defined, and we ensured consistency of variable definitions throughout this subsection. The swelling ratio is now calculated as: Swelling ratio (%)= [(W_s-W_d,re)/W_s] ×100

 

•Lines 198–209: The description of metabolite changes in Results is correct, but is primarily narrative in nature. Please emphasize that the observed trends are not always linear with the dose of Male Fig Powder.
[Response]: Lines 203–205: We thank the reviewer for catching this oversight. We agree and revised the Results text to explicitly note that several metabolites exhibited non-linear, formulation-dependent responses rather than monotonic dose-dependence.

 

•Lines 223–228: "Numerically, all FK groups showed higher mean DPPH values..." — again, a statistically insignificant effect is highlighted...
[Response]: Lines 240–241: We thank the reviewer for this important suggestion. We agree and revised the Results text to avoid overemphasizing non-significant trends. The passage now reports the numerical tendency while clearly stating that no statistically significant differences were detected among treatments (p ≥ 0.05).

 

•Lines 279–286: The caption for Figure 2 includes "PK fermentations" and "PK fermentation," which is most likely an editorial error. Please correct this to "FK fermentations" or another consistent name used in the manuscript.

[Response]: Lines 293–296: We thank the reviewer for pointing out this error. We corrected this editorial error and ensured consistent terminology throughout.

 

•Lines 320–321: “Succinate is additionally recognized as a microbiota-associated metabolite involved in host metabolic signaling…” — This fragment is a digression and does not contribute much to the interpretation of the results presented in the paper.
[Response]: Lines 334–336: We thank the reviewer for encouraging a more data-grounded interpretation. We agree and revised the statement to clearly indicate it as a possible explanation requiring further validation.

 

•Lines 322–325: “Which may reflect matrix-dependent partitioning or differences in microbial utilization…” — This is a speculative hypothesis, not supported by any additional data. Please clearly mark this as a possible explanation, not an interpretation derived directly from the experiment.
[Response]: Lines 336–340: We thank the reviewer for this helpful comment. We agree that the previous wording was overly interpretive. We revised the Discussion to describe the caffeine pattern as an observed result and to frame the explanation as a possibility, supported by literature showing that caffeine catabolism depends on specific microorganisms/enzymatic pathways and therefore may not occur in all fermentation systems.

 

•Lines 351–370: The discussion regarding KBC is interesting, but requires better comparison with previous studies on the effects of different fermentation matrices on the yield and properties of BC. The current literature is too general. The literature has already described both the effect of various herbal infusions on the production of BC films and the use of decomposed products.

[Response]: Lines 365–382: We thank the reviewer for this important and constructive suggestion. We agree and have revised the KBC discussion to include more specific comparisons with previous studies demonstrating matrix-dependent effects on BC yield and properties.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study combines compositional analysis with material characterization, which is a clear strength. The experimental design is generally sound, and the results suggest promising effects of male fig supplementation, particularly on bacterial cellulose yield.

While the fermentation setup is described clearly, the manuscript lacks important process monitoring parameters such as pH evolution, sugar consumption, or ethanol production. These variables are fundamental in kombucha fermentation and would greatly strengthen the interpretation of metabolite changes (e.g., glucuronic acid or organic acids).

Additionally, no microbiological data are provided (e.g., cell counts or population dynamics), despite using a defined culture. This makes it difficult to support conclusions related to microbial activity.

Suggestion:
Include fermentation kinetics (pH, residual sugars, ethanol if possible) and, ideally, basic microbiological data (e.g., CFU counts). At a minimum, discuss this limitation.

The manuscript occasionally implies potential health benefits, particularly regarding glucuronic acid and succinic acid. However, no biological or functional assays were performed to support such claims.

Suggestion:
Reframe these statements as compositional observations rather than functional claims or explicitly state that such implications remain hypothetical and require further validation.

Although relevant references are cited, the manuscript lacks quantitative benchmarking. For example, the reported glucuronic acid levels (up to 6.63 g/L) and BC yield (7.28 g/L) are not clearly positioned relative to existing studies.

Suggestion:
Include explicit comparisons with literature values to help readers understand whether the results represent incremental or significant improvements.

The equation provided for the swelling ratio appears identical to that used for water content, which is likely an error.

Suggestion:
Please revise and correct the formula for the swelling ratio.

The conclusions are generally well written but somewhat optimistic. It would be advisable to moderate claims regarding functionality and industrial applicability.

A brief discussion on process scalability and practical implementation would enhance the impact of the study.

Author Response

Comments and Suggestions for Authors

The study combines compositional analysis with material characterization, which is a clear strength. The experimental design is generally sound, and the results suggest promising effects of male fig supplementation, particularly on bacterial cellulose yield.

While the fermentation setup is described clearly, the manuscript lacks important process monitoring parameters such as pH evolution, sugar consumption, or ethanol production. These variables are fundamental in kombucha fermentation and would greatly strengthen the interpretation of metabolite changes (e.g., glucuronic acid or organic acids).

Additionally, no microbiological data are provided (e.g., cell counts or population dynamics), despite using a defined culture. This makes it difficult to support conclusions related to microbial activity.

 

Suggestion:
Include fermentation kinetics (pH, residual sugars, ethanol if possible) and, ideally, basic microbiological data (e.g., CFU counts). At a minimum, discuss this limitation.

The manuscript occasionally implies potential health benefits, particularly regarding glucuronic acid and succinic acid. However, no biological or functional assays were performed to support such claims.

[Response]:

Lines 393–397: We thank the reviewer for this constructive suggestion. We agree that fermentation kinetics (pH evolution, residual sugars, and ethanol) and basic microbiological monitoring (e.g., CFU counts) would strengthen mechanistic interpretation of metabolite changes. These measurements are currently being collected in ongoing follow-up experiments; however, they are beyond the intended scope of the present manuscript, which focuses on end-point compositional profiling and KBC material characterization. To avoid expanding the scope and diluting the central message, we did not include kinetic or microbiological datasets in this revision. Instead, we added an explicit limitation statement in the Discussion and noted that kinetic and CFU data will be reported in subsequent work.

 

Suggestion:
Reframe these statements as compositional observations rather than functional claims or explicitly state that such implications remain hypothetical and require further validation.

Although relevant references are cited, the manuscript lacks quantitative benchmarking. For example, the reported glucuronic acid levels (up to 6.63 g/L) and BC yield (7.28 g/L) are not clearly positioned relative to existing studies.

Include explicit comparisons with literature values to help readers understand whether the results represent incremental or significant improvements.

[Response]:

Lines 365–376: We thank the reviewer for this important and constructive comment. We agree and have revised the Discussion to avoid functional/health claims and to present glucuronic acid and succinic acid strictly as compositional outcomes of fermentation. Any potential functional relevance is now explicitly described as hypothetical and requiring dedicated biological validation beyond chemical profiling. In the same revision, we added quantitative benchmarking to position our results relative to literature values. Specifically, glucuronic acid reached 6.63 g/L in 2% FK, which exceeds the maximum value of ~2.3 g/L reported for traditional kombucha fermentation in Food Chemistry (Jayabalan et al., 2007). In addition, KBC yield reached 7.28 g/L, which is within the order of magnitude of kombucha-derived BC yields reported for nutrient-enriched/waste-derived matrices. These comparisons have been incorporated to help readers evaluate the extent of improvement achieved in the present work.

 

Suggestion:

The equation provided for the swelling ratio appears identical to that used for water content, which is likely an error.

Please revise and correct the formula for the swelling ratio.

[Response]:

Lines 168–176: We thank the reviewer for identifying this mistake. We corrected the swelling ratio equation to Swelling ratio (%)= [(W_s-W_d,re)/W_s] ×100, where W_s is the swollen mass and W_d,re is the re-dried mass.

 

Suggestion:

The conclusions are generally well written but somewhat optimistic. It would be advisable to moderate claims regarding functionality and industrial applicability.

A brief discussion on process scalability and practical implementation would enhance the impact of the study.

[Response]:

Lines 415–428: We thank the reviewer for this valuable suggestion. We agree and have revised the Conclusions to moderate the tone regarding functionality and industrial applicability, ensuring that our statements are limited to compositional and material-property outcomes supported by the present data.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors improved the work and now the manuscript is suitable for publication

Reviewer 2 Report

Comments and Suggestions for Authors

Is suitable for publication