High-Mountain Tuber Products Improve Selectively the Development and Detoxifying Capacity of Lactobacilli Strains as an Innovative Culture Strategy

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is generally clear, but there are areas where the narrative could be refined, methodology better detailed, and interpretations strengthened. Below are line-specific comments.

 

L23–26: “...without promoting the growth of pathogenic bacteria...” → specify which pathogenic species were tested.

L28–33: Results are over-condensed. Suggest including one quantitative highlight (e.g., % biofilm increase or % detoxification).

L32–33: “reported for the first time” → soften to “described for the first time to our knowledge,” to avoid overstatement.

L49–55: Citations should be more recent and specific to LAB applications in culture media. Suggest adding references beyond probiotics, e.g., LAB detoxification or environmental applications.

L66–74: This section lists many biological properties of Andean potatoes. Suggest shortening to focus on those directly relevant to LAB growth and detoxification.

L78–90: The last paragraph of the Introduction overlaps with the Abstract. Suggest rephrasing to clearly state the objectives: (1) to test phytochemical-rich tuber peel extracts as selective growth promoters of LAB, (2) to evaluate detoxification capacity of CO1.

L102–110: Provide yields in mg/g dry weight basis, not only percentages, to improve reproducibility. Clarify if extraction was done in triplicate or single batches.

L114–115: Please clarify whether L. paracasei CO1 was confirmed as non-pathogenic before assays.

L121–126: State whether pathogenic strains were handled under biosafety conditions and if ethical approvals were needed.

L157: Replicates (n=6) are mentioned later in Results. Please state here clearly.

L173: Formula for biofilm formation (%) should be double-checked; include units for clarity.

L176–180: “Specific biofilm index” is useful but not widely known. Please expand with a short rationale why it is relevant.

L189–191: Clearing zone method is semi-quantitative. Suggest including a second confirmatory assay (e.g., emulsification index, E24) for robustness.

L149–151: The sentence is duplicated. Please remove redundancy.

L152: Provide GenBank accession numbers clearly.

L249: Instead of “making it publicly available,” specify accession number (already given later).

L254–278: DNA sequence should not be included in the main text—it belongs in Supplementary Materials.

Results – LAB Growth with Extracts (L281–341)

 

L285–290: Quantitative results are good, but suggest summarizing in a table for clarity instead of long narrative.

L311–318: The explanation of dose-dependent stress is appropriate but could benefit from referencing quorum sensing literature more directly.

L355–368: Good selective activity shown. Suggest adding p-values or statistical test used (ANOVA?).

L369–376: Discussion on microbiome balance is too general. Narrow focus to how the studied extracts could realistically impact gut ecology.

L383–385: Avoid jargon such as “π→π* transitions” in Results. Move this explanation to Methods or Supplementary.

L388–390: Please quantify phenolic concentrations (mg GAE/g extract) instead of qualitative only.

L403–405: Claim “complete degradation” of phenol – indicate detection limit of methods.

L407–412: OPP degradation description is promising; clarify whether OPP metabolites were identified.

Figures captions need more detail (strain name, conditions, replicate number). Suggest reformatting Figure 2 to show statistical significance directly (asterisks are mentioned but inconsistent).

Provide deeper comparison with existing reports on LAB detoxifying phenol or other xenobiotics.

The term “symbiotic” (L333) should be replaced with “synbiotic” (standard terminology).

L499–503: Very strong claim; suggest softening to “Our results suggest...” instead of definitive “promising detoxifying activity.”

Author Response

Reviewer 1

The manuscript is generally clear, but there are areas where the narrative could be refined, methodology better detailed, and interpretations strengthened. Below are line-specific comments.

Reply: Thank you very much for taking the time to review this manuscript carefully. Your comments and valuable suggestions helped us to improve our manuscript.

 L23–26: “...without promoting the growth of pathogenic bacteria...” → specify which pathogenic species were tested.

- We agree. Names of pathogenic bacteria were included in the abstract (lines 25-27). 

L28–33: Results are over-condensed. Suggest including one quantitative highlight (e.g., % biofilm increase or % detoxification).

- In the abstract, quantitative results (% biofilm increase and % detoxification) were included in the revised manuscript (lines 30-31).

L32–33: “reported for the first time” → soften to “described for the first time to our knowledge,” to avoid overstatement.

- Regarding your comments, we write the following paragraph: “Furthermore, the bio-detoxification capacity of phenol and o-phenyl phenol, particularly of the novel strain CO1-LVP105, along with its mode of action and genetic identification is described for the first time to our knowledge” (please see in the abstract, lines 33-35).

L49–55: Citations should be more recent and specific to LAB applications in culture media. Suggest adding references beyond probiotics, e.g., LAB detoxification or environmental applications.

- We agree. A short text has been added to the introduction of a current article (page 2, lines 58-63.

[6] Petrova, et al. 2022. The complex role of lactic acid bacteria in food detoxification. Nutrients 14, 2038.

L66–74: This section lists many biological properties of Andean potatoes. Suggest shortening to focus on those directly relevant to LAB growth and detoxification.

- This suggestion has been taken into account, and the biological properties of the Andean potato have been shortened (page 2).

L78–90: The last paragraph of the Introduction overlaps with the Abstract. Suggest rephrasing to clearly state the objectives: (1) to test phytochemical-rich tuber peel extracts as selective growth promoters of LAB, (2) to evaluate detoxification capacity of CO1.

- We agree, the last paragraph of the Introduction was improved according to your suggestion (page 2, lines 86-90).

L102–110: Provide yields in mg/g dry weight basis, not only percentages, to improve reproducibility. Clarify if extraction was done in triplicate or single batchs.es.

- Yields in mg/g of dry peels were included in Table 1A (page 11). All extractions were done in single batch.

L114–115: Please clarify whether L. paracasei CO1 was confirmed as non-pathogenic before assays.

- In the revised manuscript, after the last paragraph of section 3.8 (page 15) we added the following:

“The safety profile of Lacticaseibacillus paracasei CO1-LVP105 is supported by comparison with other strains of the same species. For instance, L. paracasei strain F-19e has been evaluated under GRAS Notice No. 810 by the U.S. Food & Drug Administration, including whole-genome analyses, absence of virulence and antibiotic resistance genes, and human feeding studies with infants, children, and adults, without adverse outcomes [43]. Likewise, L. paracasei subsp. paracasei Q-1 , isolated from cow dung, showed no hemolytic, gelatinase, or mucin-degrading activity in vitro, and in vivo toxicity studies in mice (14-day acute and 28-day sub-chronic) revealed no effects on organ indices or histology [44]. These data contextualize the origin of CO1-LVP105 from healthy animals and its lack of observed pathogenic traits under our experimental conditions, although whole-genome sequencing of CO1-LVP105 remains a planned future work to confirm its safety more precisely”.

43. Arla Foods Ingredients Group P/S & Heimbach, J. T. GRAS Notice No. 810: Lactobacillus paracasei subsp. paracasei strain F-19e - intended use in powdered milk-based infant formula; scientific procedures. U.S. Food & Drug Administration 2019. Retrieved from https://www.fda.gov/media/129477/download
44. Wang, F.;Chen, L.; Li, Y.; Gao, G.; Wang, Y.; Shi, R.; Zhang, H.; Chen, Y.; Cao, R.; Shi, Q.; Wu, T. (). Lacticaseibacillus paracasei subsp. paracasei Q-1 exhibits good safety and effectively prevents Escherichia coli K99-induced diarrhea in mice. Probiotics Antimicrob. Proteins 2025. Advance online publication. https://doi.org/10.1007/s12602-025-10552-z

L121–126: State whether pathogenic strains were handled under biosafety conditions and if ethical approvals were needed.

- We appreciate this comment. All pathogenic bacterial strains (Escherichia coli ATCC 35218, E. coli O157:H12, Salmonella enterica serovar Typhimurium ATCC 14028, S. corvalis SF2 and S. cerro SF16) were handled in accordance with standard biosafety procedures. Experiments were conducted in a Biosafety Level 2 (BSL-2) laboratory at the Faculty of Biochemistry, Chemistry and Pharmacy (Universidad Nacional de Tucumán), using certified containment facilities and following institutional guidelines. Because only bacterial strains were used and no animals or human participants were involved, no ethical approval was required under current national and institutional regulations. We have now included this statement in the revised manuscript (Materials and Methods, page 3, lines 130-134).

L157: Replicates (n=6) are mentioned later in Results. Please state here clearly.

- We agree. These replicates (n=6, in two independent experiment) were clearly indicated in the experimental section (page 4, line 159).

L173: Formula for biofilm formation (%) should be double-checked; include units for clarity.

- Thank you for this observation. We have double-checked the calculation and confirmed that the correct expression is:

Biofilm formation (%) = (As / A0) × 100, where: A0 is the absorbance of the control wells (biofilm biomass in untreated medium, measured at 560 nm) and As is the absorbance of each sample (biofilm biomass under test conditions, measured at 560 nm). The result is expressed as a percentage (%) of the control. This clarification has been added to the revised manuscript (Materials and Methods, page 4, line 175).

L176–180: “Specific biofilm index” is useful but not widely known. Please expand with a short rationale why it is relevant.

- In the experimental section (page 4, lines 186-187) we write: “The degree of biofilm production is classified in three categories according to this useful index: weak (SBI ≤ 0.5), moderate (0.5 > SBI ≤ 1), and strong (SBI > 1) [16].

16- Martinez-Medina, M.; Naves, P.; Blanco, J.; Aldeguer, X.; Blanco, J.E.; Blanco, M., Ponte, C.; Soriano, F.; Darfeuille-Michaud, A.; Garcia-Gil, L.J. Biofilm formation as a novel phenotypic feature of adherent-invasive Escherichia coli (AIEC). BMC Microbiol. 2009, 9, 202.

L189–191: Clearing zone method is semi-quantitative. Suggest including a second confirmatory assay (e.g., emulsification index, E24) for robustness. 

- In Table 3 (LAB surfactant and emulsifying activities), emulsification index was added (page 15).

L149–151: The sentence is duplicated. Please remove redundancy.

- This sentence was deleted (page 4).

L152: Provide GenBank accession numbers clearly.

- The following sentence was added:  “The 16S ribosomal DNA sequence of the isolated strain was deposited in the GenBank, Accession number: PV763827” (page 4, line 157).

L249: Instead of “making it publicly available,” specify accession number (already given later).

- This sequence was deposited in GenBank, Accession number: PV763827. The isolated strain Lacticaseibacillus paracasei was lyophilized and incorporated into the LIVAPRA strain collection with the CO1-LVP105 (page 7, lines 291-293).

L254–278: DNA sequence should not be included in the main text—it belongs in Supplementary Materials.

- DNA sequence has been included in Supplementary material.

Results – LAB Growth with Extracts (L281–341)

 L285–290: Quantitative results are good, but suggest summarizing in a table for clarity instead of long narrative.

- The results of the extracts on pathogenic bacteria are summarized in a table (supplementary material), while the results on non-pathogenic bacteria are presented in figures, which we unfortunately cannot modify for reasons of time.

L311–318: The explanation of dose-dependent stress is appropriate but could benefit from referencing quorum sensing literature more directly.

- We agree. This connection was made directly (pages 8-9).

L355–368: Good selective activity shown. Suggest adding p-values or statistical test used (ANOVA?).

- We agree. Tukey’s test was used.

L369–376: Discussion on microbiome balance is too general. Narrow focus to how the studied extracts could realistically impact gut ecology

- We thank the reviewer for this valuable suggestion. The following paragraph has been included in the discussion: “The polyphenolic-rich extracts from Andean tuber peels evaluated in this study significantly enhanced the growth and biofilm formation of Lactiplantibacillus plantarum ATCC 10241 and Lacticaseibacillus paracasei CO1-LVP105, while not stimulating and in some cases reducing biofilm formation of Gram-negative enteric pathogens. Given their composition (flavonoids, ferulic acid, coumarins), these extracts could act as selective prebiotic substrates that favor colonization and persistence of beneficial lactobacilli while limiting pathogen adhesion. This selective stimulation, combined with the biosurfactant and detoxifying activities exhibited by the studied LAB strains, would help beneficial bacteria occupy intestinal niches, strengthen mucosal barriers, and competitively exclude pathogens. Such a mechanism provides a plausible and realistic way in which these extracts could support gut ecology and host health. Previous studies have also reported selective antibacterial and antipathogenic effects of diverse plant-derived compounds, which supports our findings [20,35]”(pages 9-10).

L383–385: Avoid jargon such as “π→π* transitions” in Results. Move this explanation to Methods or Supplementary.

- These transitions were clarified as “electronic transitions” (page 10).

L388–390: Please quantify phenolic concentrations (mg GAE/g extract) instead of qualitative only.

- In the bioguided study, the Table 1B (Quantification of Phenolic Compounds of O. tuberosa var. oca rosa) was included (page 12).

L403–405: Claim “complete degradation” of phenol – indicate detection limit of methods.

- The word complete was deleted, and the estimated sensitivity limits of the methods used are indicated (page 12).

L407–412: OPP degradation description is promising; clarify whether OPP metabolites were identified.

- OPP degradation metabolites were not identified by GC-MS.

Figures captions need more detail (strain name, conditions, replicate number). Suggest reformatting Figure 2 to show statistical significance directly (asterisks are mentioned but inconsistent).

- Figure 1 and 2 captions were completed, and asterisks indicate significance in the revised manuscript (pages 7-8).

Provide deeper comparison with existing reports on LAB detoxifying phenol or other xenobiotics.

- Other antecedents were included (page 12).

The term “symbiotic” (L333) should be replaced with “synbiotic” (standard terminology).

- We agree. This change was made (page 9).

 L499–503: Very strong claim; suggest softening to “Our results suggest...” instead of definitive “promising detoxifying activity.”

- Thank you very much for this suggestion. This sentence was modified accordingly (page 16).

Reviewer 2 Report

Comments and Suggestions for Authors

Comments are included in the file attached

Comments for author File: Comments.pdf

Author Response

Reviewer 2

After carefully review the manuscript untitled: “Innovative culture media designed with high-mountain tuber peel extracts improve selectively the development and detoxifying capacity of lactobacilli strains” the decision is accepted after minor revisions. The manuscript provides novel insights of firstly identify Lacticaseibacillus paracasei CO1. Additionally, the effect of selected high-mountain potato peel extracts on this LAB were remarkable and brought new knowledge into this field of study.

 Reply

All authors thank your comments of our manuscript, which was improved based on your valuable suggestions.

 Comments and questions:

• Please, include highlighting results (numeric) of your work in the abstract.

- We agree. Highlighting results were added.

• Please, be more concise with keywords. The current keywords are too long. Try to choose short, specific and valuable keywords.

- Yes, we agree; the keywords were changed and highlighted in yellow.

• The text between lines 149-151 must be removed because it is repeated.

-  We agree; these repeated lines have been removed (page 4).

• A great number of lactic acid bacteria are able to produce extracellular or/and cell bound (CB) biosurfactants. Did the authors check if these LAB strains are also CB biosurfactant producers? If yes, the results must be included or at least mentioned and discussed.

- In this study, we evaluated the LAB biosurfactants released into the culture medium. We appreciate the valuable information provided, which we consider interesting to address in future studies. Nevertheless, this topic has been included in the discussion through the following paragraph:

“A great number of bacteria are able to produce extracellular or/and cell bound biosurfactants [40,41]. Indeed, Lara and co-authors (2025) identified a LAB strain, Lactiplantibacillus plantarum Tw226, as a cell-bound surfactant producer. This biosurfactant has a glycolipopeptide nature and is useful as an emulsifier of food ingredients such as corn oil. Studies on biosurfactants produced by LAB as essential oils' emulsifiers are quite scarce [41]. Therefore, its approach in L. paracasei CO1-LVP105, as well as studies of functional and safety properties would be essential in future studies” (page 15).

 

40. Lvova, K.; Martínez-Arcos, A.; López-Prieto, A.; Vecino, X.; Moldes, A.B.; Cruz, J.M. Optimization of the operational conditions to produce extracellular and cell-bound biosurfactants by Aneurinibacillus aneurinilyticus using corn steep liquor as a unique source of nutrients. Fermentation 2023, 9, 351.
41. Lara, V.M.; Gliemmo, M.F.; Vallejo, M.; García González, M.d.C.; Alfaro Rodríguez, M.d.C.; Campos, C.A. Use of the glycolipopeptid biosurfactant produced by Lactiplantibacillus plantarum Tw226 to formulate functional Cinnamon bark essential oil emulsions. Foods 2025, 14, 1540.

Reviewer 3 Report

Comments and Suggestions for Authors

Fermentation-3826474

The manuscript examines the effects of thirty-two extracts derived from various tuber varieties (potatoes) from the Argentinean Puna on two strains of lactic acid bacteria: Lactiplantibacillus plantarum ATCC 10241 (a food-origin probiotic bacterium) and Lacticaseibacillus paracasei CO1 (from ovine origin, for which identification data are also provided).  The effects tested included bacterial growth, biofilm quantification, biofilm index, surfactant activity, and polyphenol oxidase, using culture media added with increasing concentrations of extracts.

The topic may be relevant since the waste from these tubers could reach substantial amounts, and their revalorization could provide essential revenues. However, information on their composition and possible functional properties is scarce in the literature. For the assays, habitual culture media are used, added with increasing concentrations of the diverse extracts; however, the objective is not the development of new culture media but the estimation of the effects of the added extracts. Following the discussion of the results, the conclusion is relatively weak and could be improved. Finally, the manuscript is supported by a sufficient number of references.

It was also expected that more detailed information on the composition of the extracts would be provided, as the use of GC-MS should have provided more detailed information on the specific compounds included in each extract. Authors are encouraged to add such a composition in light of the already conducted analysis.

Specific comments

Title. According to the manuscript text, the objective was not the development of a new culture, but to study the effect of the added extracts on the growth and other properties of the two tested lactic acid bacteria. In the reviewer's opinion, the title should be revised to align with the objective of the assays. Additionally, lines 78-79 should also be modified and the text revised accordingly.

Abstract. It is excessively detailed regarding the origins of the extracts but reduced with respect to the results. Adding relevant information about the effects could be helpful for readers who access the abstract exclusively. Line 32, the sentence is excessively inclusive, providing that only the mutagenic compound tested was phenol.

Introduction

Line 43 provides information on the meaning of LAB. The acronym could already be used later in lines 45-46.

Line 78-90. Please rewrite this section to be more precise regarding the objective of the work. Remove also information of results not appropriate (line 87-88)

Materials and methods

Lines 103-105 and text in general. Please, revise the use of italics in scientific names of species.

Line 123-125. The information regarding external contributions is usually included in the Acknowledgments section at the end of the text

Lines 149-151. The text is duplicated.

Line 156. The sentence could be simplified.

Line 158. Please include the full name first use of DMSO.

Line  165-166. The adaptations should be included.

Line 168. Simplify

Lines 171-174. The sentence could be simpler.

Line 178. Remove “in our case”

Line 230. Include the full names of PhOH and OPP

Results and Discussion

Line 254-278. The information in section 3.1.1 could be provided as supplementary material.

Line 282-284. This sentence hardly aligns with the subsequent information. Something is lost or needs modification.

Line 295-296 and supplementary material. Usually, asterisks (* to ***) are used to denote significance, not as applied in this case. Also include the names of the tested effects in the legend. “Effects on growth and biofilm formation of ……”

L311-318. Please clarify the paragraph.

Lines 323-324. See the above comment

Lines 338-340. Please comment on this trend in relation to those observed in this work.

Figure 3. The usefulness of this figure is doubtful. Additionally, the transfer of its interpretation to readers is questionable. What should they revise? In the event of continued use, its usefulness should be justified in the text and legend, with relevant information included to enhance understanding.

Conclusion

As previously commented, it should also include additional relevant information regarding the most outstanding results.

Author Response

Reviewer 3

The manuscript examines the effects of thirty-two extracts derived from various tuber varieties (potatoes) from the Argentinean Puna on two strains of lactic acid bacteria: Lactiplantibacillus plantarum ATCC 10241 (a food-origin probiotic bacterium) and Lacticaseibacillus paracasei CO1 (from ovine origin, for which identification data are also provided). The effects tested included bacterial growth, biofilm quantification, biofilm index, surfactant activity, and polyphenol oxidase, using culture media added with increasing concentrations of extracts.

The topic may be relevant since the waste from these tubers could reach substantial amounts, and their revalorization could provide essential revenues.

However, information on their composition and possible functional properties is scarce in the literature. For the assays, habitual culture media are used, added with increasing concentrations of the diverse extracts; however, the objective is not the development of new culture media but the estimation of the effects of the added extracts. Following the discussion of the results, the conclusion is relatively weak and could be improved.

Finally, the manuscript is supported by a sufficient number of references.

It was also expected that more detailed information on the composition of the extracts would be provided, as the use of GC-MS should have provided more detailed information on the specific compounds included in each extract. Authors are encouraged to add such a composition in light of the already conducted analysis.

 

Reply

Thank you very much for taking the time to review this manuscript carefully. Your comments and valuable suggestions helped us to improve our manuscript.

In response to your concern, our study was bio-guided, and the most active extracts were selected for chemical characterization through routine phytochemical and spectroscopic testing, with an emphasis on microbiological studies. GC-MS of the Oxalis tuberosa subextract, selected for its activity, revealed one main compound (terpenoid), and minor compounds (unidentified). Phenolic compounds determined in the preliminary phytochemical analysis were not detected by GC-MS due to their non-volatile nature; therefore, we decided that GC-MS should not be included in the manuscript. HPLC-MS-MS is planned to continue the chemical study with greater specificity in the near future (this technology is not available in our laboratory or at our university.).

 

Title: According to the manuscript text, the objective was not the development of a new culture, but to study the effect of the added extracts on the growth and other properties of the two tested lactic acid bacteria. In the reviewer's opinion, the title should be revised to align with the objective of the assays. Additionally, lines 78-79 should also be modified and the text revised accordingly.

- We agree; the title, objective, and the full text have been revised according to your appropriate observation.

Abstract: It is excessively detailed regarding the origins of the extracts but reduced with respect to the results. Adding relevant information about the effects could be helpful for readers who access the abstract exclusively. Line 32, the sentence is excessively inclusive, providing that only the mutagenic compound tested was phenol.

- These changes have been made to the new version of our manuscript, thanks to your valuable help.

Introduction

Line 43 provides information on the meaning of LAB. The acronym could already be used later in lines 45-46.

- We agree. The acronym LAB was used in lines 48 and 52 (page 2, in the revised manuscript), as throughout the manuscript, after the full name and acronym were mentioned for the first time (line 45).

Line 78-90. Please rewrite this section to be more precise regarding the objective of the work. Remove also information of results not appropriate (line 87-88).

- This section has been improved based on your suggestions (page 2, lines 87-90).

Materials and methods

Lines 103-105 and text in general. Please, revise the use of italics in scientific names of species.

- We agree. All scientific names of species are now written in italics.

Line 123-125. The information regarding external contributions is usually included in the Acknowledgments section at the end of the text.

- This information was included in the Acknowledgments section in the revised manuscript.

Lines 149-151. The text is duplicated.

- The duplicate paragraph has now been removed (page 4).

Line 156. The sentence could be simplified.

- We agree, please revise the sentence highlighted in yellow (page 4, lines 159-160).

Line 158. Please include the full name first use of DMSO.

- The full name was included in line 161 (page 4).

Lines 165-166. The adaptations should be included.

- The text has been modified as follows: “Crystal violet (CV) assay was used to quantify the development of biofilm on a polystyrene microplate, which was based on a protocol previously published [15]. The micro-method implemented allowed the use of small volumes (µL) and amounts of samples (µg), and a 0.1% solution of CV was employed” (page 4, lines 169-170).

Line 168. Simplify.

- We agree.

Lines 171-174. The sentence could be simpler.

- We agree. This sentence has been modified.

Line 178. Remove “in our case”.

- In line 167, “in our case” was deleted (page 4, line 182).

Line 230. Include the full names of PhOH and OPP.

- Now, in the revised manuscript, the PhOH and OPP full names were included (page 6 lines 251 and 264).

Results and Discussion

Line 254-278. The information in section 3.1.1 could be provided as supplementary material.

- The information in section 3.1.1 has been provided as supplementary material in the new version of our manuscript.

Line 282-284. This sentence hardly aligns with the subsequent information. Something is lost or needs modification.

- We agree; this sentence was modified and highlighted in yellow (page 7, lines 277-299).

Line 295-296 and supplementary material. Usually, asterisks (* to ***) are used to denote significance, not as applied in this case. Also include the names of the tested effects in the legend. “Effects on growth and biofilm formation of ……”

- Thank you for pointing this out. We agree with this comment. Therefore, we have included the names of the effects in the legends, and asterisks were now used indicating significance.

L311-318. Please clarify the paragraph.

- This paragraph has been modified (pages 8).

Lines 323-324. See the above comment

Lines 338-340. Please comment on this trend in relation to those observed in this work.

- Thank you for pointing this out. We have expanded the sentence to explicitly relate our own findings to this trend. In our study we also observed that, for certain extracts and concentrations, biofilm stimulation occurred without a parallel increase in planktonic growth. This pattern is consistent with an adaptive response to chemical stress mediated by quorum sensing, as described by Verni et al. [20], and suggests that exposure to specific polyphenolic compounds can trigger a biofilm-forming phenotype without necessarily promoting cell division. We have added this explanation in the revised manuscript (page 9).

Figure 3. The usefulness of this figure is doubtful. Additionally, the transfer of its interpretation to readers is questionable. What should they revise? In the event of continued use, its usefulness should be justified in the text and legend, with relevant information included to enhance understanding.

- We agree; this figure has been replaced by a table for a clearer understanding. A table containing the retention times and area of the OPP peaks obtained in the different biodegradation treatments, as well as a comparative image of the OPP peaks (magnified) and mass spectrum, was included in the revised version of the manuscript (page 13).

 Conclusion

As previously commented, it should also include additional relevant information regarding the most outstanding results.

- The Conclusions section has been revised to include additional relevant information (page 16).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have made the suggested modifications and the paper is now improved, I have no further comments.