Enzymatic hydrolysis of starch from the anthocyanin extraction residue (AER-starch) with ultrasound pretreatment: A techno-economic assessment

J.D. Quiñonez-Ensuncho; Susana Ochoa; Víctor-Manuel Osorio-Echeverri; J. Felipe Osorio-Tobón

doi:10.1371/journal.pone.0343968

Peer Review History

Original SubmissionNovember 7, 2025
9 Dec 2025 Decision Letter - Leonidas Matsakas, Editor Dear Dr. Osorio-Tobón, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jan 23 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Leonidas Matsakas Academic Editor PLOS One Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please note that PLOS One has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. In these cases, we expect all author-generated code to be made available without restrictions upon publication of the work. Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse. 3. Thank you for stating the following financial disclosure: “Institución Universitaria Colegio Mayor de Antioquía. Convocatoria de investigación. FCSA23.” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 4. Thank you for stating the following in the Acknowledgments Section of your manuscript: “The authors are grateful to COLMAYOR for financial support.” We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “Institución Universitaria Colegio Mayor de Antioquía. Convocatoria de investigación. FCSA23.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 5. We note that your Data Availability Statement is currently as follows: All relevant data are within the manuscript and its Supporting Information files. Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition). For example, authors should submit the following data: - The values behind the means, standard deviations and other measures reported; - The values used to build graphs; - The points extracted from images for analysis. Authors do not need to submit their entire data set if only a portion of the data was used in the reported study. If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access. 6. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 7. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: I Don't Know ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: No ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: The manuscript explores ultrasound pretreatment to enhance enzymatic hydrolysis of starch recovered from purple yam extraction residues. consecutive enzymatic hydrolysis (CEH) and simultaneous enzymatic hydrolysis (SEH) were tested with and without ultrasound, and hydrolysis degree, starch conversion, and techno economic feasibility were evaluated using SuperPro Designer. The authors report that ultrasound improves hydrolysis at low starch concentrations, CEH outperforms SEH, and economic viability depends on feedstock cost and glucose syrup price. Although the work is relevant and integrates experimental and economic analysis, the manuscript needs major revision to improve clarity, methodological detail, interpretation consistency, and transparency of the economic assessment. Review comments: • The manuscript would benefit from a clearer mechanistic explanation of the ultrasound pretreatment, particularly at higher starch concentrations, to strengthen the scientific rationale presented in the Introduction section. • A more detailed discussion of the observed combination of high SC and lower HD would enrich the understanding of potential oligosaccharide formation and saccharification completeness. • Sections 2.2 & 2.3, Including thermal-only controls and confirming enzyme stability under SEH conditions would enhance the robustness of the experimental design and improve interpretation of ultrasound-specific effects. • Section 2.3, Reporting additional ultrasound parameters such as delivered power, acoustic energy density, and temperature control would support reproducibility and alignment with literature. • Section 3.1, Providing a more detailed explanation for cases where SC exceeds 90% but HD remains lower would enrich understanding of oligosaccharide formation and saccharification completeness. • Sections 3.1 & 3.2, Clarifying the varying influence of ultrasound across experiments would help present a more cohesive interpretation, as SEH and CEH results differ in several places. • Including mass and energy balance summaries would support validation of the process model and clarify how each unit operation contributes to overall process performance. • In Techno Economic Evaluation (Section 2.6), Providing justification for key assumptions such as labor cost, resin replacement rates, evaporation energy, and depreciation, would improve transparency of the economic modeling. • In the TEA Results and Sensitivity Analysis, supporting assumed AER starch prices with market references would make the economic conclusions more robust and credible. • Discussing scale-up factors such as mixing, heat transfer, enzyme kinetics, and viscosity would improve the realism of extrapolating laboratory performance to industrial conditions. • Expanding the literature review to include recent advances in ultrasound-assisted enzymatic hydrolysis and cavitation mechanisms would enhance the scientific context in the Introduction section. • Including a brief comparison with existing commercial glucose syrup production routes could further strengthen the TEA by highlighting the competitive position and market potential of glucose syrup derived from AER starch. • Incorporating a short discussion on operational risks, such as feedstock variability or potential equipment downtime, would add practical context and help readers appreciate real-world implementation factors. Reviewer #2: The manuscript is well organized and documents all experimental protocols. However, the process modelling and technoeconomic assessment (TEA) lack standard documentation of modelling and economic assumptions, which makes it difficult to follow the TEA results. I suggest the authors consider revising the following aspects: Section 2.5 Expand/clarify process modeling details. A paragraph can be added to describe the following: - Thermodynamic/physical property methods used in SuperPro Designer - How non-conventional components such as AER-starch and protiens were represented. Pseudo-components or user defined? - Whether full energy balances were solved, how electricity/steam demands were estimated for the evaporation, filtration and mixing steps Section 3.3 Strengthen the justification for excluding ultrasound in the TEA flowsheet. It was mentioned that ultrasound at high starch concentration does not significantly improve performance and would increase COM; however, in the results it was stated only that ultrasound was not considered further. You could: - Include a simplified alternative flowsheet with ultrasound units (even if only described qualitatively). - Provide a short comparative COM estimate (e.g., “preliminary analysis indicated that including ultrasound increased COM by X–Y% at 350 g·L⁻¹ with no significant yield benefit”). - Clearly state why ultrasound is attractive at low concentrations (process intensification) but not at the industrially relevant high concentrations. Section 3.4 Clarify the economic framework more systematically. Right now, elements like discount rate (7%), payback, ROI, IRR, and NPV are described in the results, but not all main cash-flow assumptions are grouped in one place. I’d suggest to add a small “Economic assumptions” table/box containing: - Project lifetime (years). - Discount rate. - Tax rate (or explicitly state that taxes are neglected). - Depreciation method and period. - Working capital assumptions (if any). - Currency and reference year for all costs (e.g., “all costs in 2024 USD”). - Basis (330 days/year, 5000 L batch, plant availability, etc.). That makes the TEA more standard and easier to compare with other studies. ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: No Reviewer #2: Yes: Sennai Mesfun ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. https://doi.org/10.1371/journal.pone.0343968.r001
Revision 1
15 Jan 2026 Author Response Journal Requirements: The reply and manuscript’s text adjustments of Reviewer #1 comments were highlighted in green. We thank the academic editor and PLOS ONE staff thorough review of the manuscript. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Reply: Thank you for your feedback. We have reviewed the style requirements. 2. Please note that PLOS One has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. In these cases, we expect all author-generated code to be made available without restrictions upon publication of the work. Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse. Reply: Thank you for your feedback. All relevant data are provided within the manuscript. The process simulations were performed using SuperPro Designer v14®, a commercially licensed software. No author-generated code was used in this study. As the software is proprietary, it cannot be shared. However, all model parameters, assumptions, and input data required to reproduce the simulations have been fully described and are available in the manuscript. 3. Thank you for stating the following financial disclosure: “Institución Universitaria Colegio Mayor de Antioquía. Convocatoria de investigación. FCSA23.” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. Reply: Thank you for your feedback. We have reviewed it. 4. Thank you for stating the following in the Acknowledgments Section of your manuscript: “The authors are grateful to COLMAYOR for financial support.” We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “Institución Universitaria Colegio Mayor de Antioquía. Convocatoria de investigación. FCSA23.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Reply: Thank you for your feedback. We have reviewed it. 5. We note that your Data Availability Statement is currently as follows: All relevant data are within the manuscript and its Supporting Information files. Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition). For example, authors should submit the following data: - The values behind the means, standard deviations and other measures reported; - The values used to build graphs; - The points extracted from images for analysis. Authors do not need to submit their entire data set if only a portion of the data was used in the reported study. If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access. Reply: Thank you for your feedback. We confirm that all raw data required to replicate the results of our study have been made publicly available. The complete dataset is openly accessible on Zenodo at the following DOI: https://doi.org/10.5281/zenodo.18257051 6. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. Reply: Thank you for your comment. We have revised the manuscript accordingly. The phrase “data not shown” has been removed and all data have now been made publicly available. These data are included in the dataset deposited in Zenodo, accessible at the following DOI: https://doi.org/10.5281/zenodo.17872033 7. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. Reply: Thank you for your feedback. We have reviewed it. Reviewers' comments: Reviewer #1: The reply and manuscript’s text adjustments of Reviewer #1 comments were highlighted in yellow. We thank the Reviewer for the thorough review of the manuscript. 1. The manuscript would benefit from a clearer mechanistic explanation of the ultrasound pretreatment, particularly at higher starch concentrations, to strengthen the scientific rationale presented in the Introduction section. Reply: Thank you for your feedback. We have added the following paragraph to the discussion: At higher starch concentrations, the solution viscosity increased, diminishing the performance of the ultrasound pretreatment. The increase in the starch solution viscosity limits the bubble growth and collapse, leading to a significant rise in the cavitation threshold [27]. Although higher amplitudes can partially compensate for the effect of high viscosity, amplitudes above 60% could not be used in this study to preserve probe integrity and ensure equipment maintenance. Consequently, ultrasound pretreatment becomes less effective at higher AER-starch concentrations. 2. A more detailed discussion of the observed combination of high SC and lower HD would enrich the understanding of potential oligosaccharide formation and saccharification completeness. Reply: Thank you for your feedback. We have added the following paragraph to the discussion: In general, shorter hydrolysis times resulted in higher SC values but lower HD values, suggesting the accumulation of partially hydrolyzed oligosaccharides. In this study, although longer saccharification times increased HD values, complete saccharification to glucose was not achieved, and the positive effects of the ultrasound pretreatment were limited. Under the conditions studied, starch accessibility was unlikely to have been the limiting factor, and the saccharification step determined hydrolysis completeness. Overall, ultrasound at higher AER-starch concentrations had a limited impact on HD and no effect on SC. Therefore, the ultrasound pretreatment does not improve the hydrolysis at high AER-starch concentrations. 3. Sections 2.2 & 2.3, Including thermal-only controls and confirming enzyme stability under SEH conditions would enhance the robustness of the experimental design and improve interpretation of ultrasound-specific effects. Reply: Thank you for this observation. The SEH was explored as a strategy to reduce overall processing time by combining the liquefaction and saccharification steps. However, the hydrolysis yields obtained under SEH conditions were lower than those achieved in the sequential process. This behavior can be explained by the different optimal temperature requirements of the enzymes employed. The α-amylase used is an industrial enzyme designed to operate at high temperatures (90 °C), whereas amyloglucosidase exhibits optimal activity at lower temperatures (60 °C). Operating SEH at an intermediate temperature (75 °C) represented a compromise that likely reduced amyloglucosidase activity and limited saccharification efficiency as we mentioned in the text. These results highlight the importance of enzyme thermal compatibility when implementing SEH, rather than indicating intrinsic limitations of the process or enzyme performance. 4. Section 2.3, Reporting additional ultrasound parameters such as delivered power, acoustic energy density, and temperature control would support reproducibility and alignment with literature. Reply: Thank you for your feedback. We have added the following information to the manuscript: Ultrasound pretreatment was performed using a 750 W ultrasonic homogenizer (Cole-Parmer, Vernon Hills, IL, USA) equipped with temperature control (30°C). AER-starch mixtures were treated for 30 minutes at an amplitude of 60% in pulse mode (2 seconds ON, 2 seconds OFF), with an estimated delivered power of 600 W and an acoustic energy density of 592 W cm-². 5. Section 3.1, Providing a more detailed explanation for cases where SC exceeds 90% but HD remains lower would enrich understanding of oligosaccharide formation and saccharification completeness. Reply: Thank you for your feedback. We have added the following information to the manuscript: The combination of higher SC values (>90%) and lower HD values, observed mainly in the simultaneous process, can be explained by the formation and accumulation of soluble oligosaccharides rather than by complete saccharification to glucose. SC reflects the solubilization of AER-starch into soluble compounds, whereas HD (determined by the DNS method) quantifies reducing sugars such as glucose and short-chain saccharides. In CEH and UCEH, higher SC and SC values were obtained, indicating that starch solubilization followed by saccharification was an effective process. In contrast, lower values of SC and HD were obtained in SEH because the hydrolysis was performed at a temperature outside the optimal range recommended by the enzyme supplier. Although ultrasound pretreatment enhances starch solubilization, it did not significantly promote the enzymatic conversion of soluble compounds into glucose under simultaneous hydrolysis conditions. Thus, while disruption of starch granules and generation of soluble intermediate compounds are effective during pretreatment and liquefaction, saccharification completeness is governed by temperature and pH conditions rather than starch accessibility alone. 6. Sections 3.1 & 3.2, Clarifying the varying influence of ultrasound across experiments would help present a more cohesive interpretation, as SEH and CEH results differ in several places. Reply: Thank you for your feedback. We agree that clarifying the differing influence of ultrasound across CEH and SEH is important for a cohesive interpretation of the results. Based on your suggestion and related comments from the other reviewer, we revised Sections 3.1 and 3.2 to explicitly distinguish the role of ultrasound under each hydrolysis strategy. 7. Including mass and energy balance summaries would support validation of the process model and clarify how each unit operation contributes to overall process performance. Reply: Thank you for the comment. This information has been compiled and is now presented as supplementary material in S2 Table. 8. In Techno Economic Evaluation (Section 2.6), Providing justification for key assumptions such as labor cost, resin replacement rates, evaporation energy, and depreciation, would improve transparency of the economic modeling. Reply: Thank you for the comment. This information has been compiled and is now presented as supplementary material in S1 Table. 9. In the TEA Results and Sensitivity Analysis, supporting assumed AER starch prices with market references would make the economic conclusions more robust and credible. Reply: Thank you for your feedback. In the market, AER-starch is not commercially available on the market. Thus, its purchasing cost was estimated based on the price of cassava starch, which can be considered a comparable product. In this context, the price was obtained from a local grocery store. We have added the market reference to the manuscript. 10. Discussing scale-up factors such as mixing, heat transfer, enzyme kinetics, and viscosity would improve the realism of extrapolating laboratory performance to industrial conditions. Reply: Thank you for your feedback. We have added the following information to the manuscript: Factors such as mixing and heat transfer, as well as viscosity and enzyme kinetics, should be considered during scale-up. For example, an increase in viscosity can reduce bubble collapse, reducing the cavitation effect and mass transfer rates. Moreover, the distribution of ultrasound energy is influenced by reactor size because cavitation is not uniform across the reactor. Moreover, an increase in temperature requires precise temperature control to preserve enzyme activity [18]. On the other hand, as acoustic cavitation may negatively affect enzyme stability, the ultrasound is commonly applied as a pretreatment rather than simultaneously. In practice, perfect mixing conditions cannot be achieved at a large scale, and there is no single consensus approach for scale-up [19]. Instead, a scale-up approach could rely on maintaining comparable laboratory-scale operating conditions and assuming they can be extrapolated, while acknowledging unavoidable gradients in temperature, pH, and concentration at the industrial scale. In this approach, we assumed that the HD and SC of the hydrolysates obtained at the laboratory scale would be reproduced at larger scales under the same hydrolysis conditions (e.g., AER-starch concentration, hydrolysis time, temperature, etc.). 11. Expanding the literature review to include recent advances in ultrasound-assisted enzymatic hydrolysis and cavitation mechanisms would enhance the scientific context in the Introduction section. Reply: Thank you for your feedback. We have added the following information to the manuscript: Depending on the matrix and enzyme, the ultrasound operation parameters have different effects. For example, during the preparation of hydrolyzed egg yolk powder after ultrasound pretreatment, cavitation and excessive ultrasound led to gradual increases and decreases in surface hydrophobicity and free sulfhydryl groups, as observed with increasing ultrasound time. In consequence, the protein swollen with disulfide bonds becomes more aggregated, and the reconstruction of these bonds was facilitated by cavitation [11]. Cavitation also generates microjets and radicals, thereby increasing selective disruption of the cell wall in lignoc Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0343968.r002
4 Feb 2026 Decision Letter - Leonidas Matsakas, Editor Dear Dr. Osorio-Tobón, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Mar 21 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Leonidas Matsakas Academic Editor PLOS One Journal Requirements: If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: N/A ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: The revision has addressed the main concerns and substantially improved clarity and TEA transparency. However, minor revisions are required before the manuscript can be accepted. The remaining points are as follows: • Please standardize units/notation (e.g., g/L, kg/kg, US$/kg) and define all acronyms at first mention (AER, SC, HD, CEH/SEH/UCEH) to improve readability. • Please add a brief justification (or citation) for the selected enzyme dosages and hydrolysis duration to strengthen comparability with prior studies. • Please clarify the intended syrup product specification (e.g., target solids content and typical sugar profile) so the selling-price assumption is easier to interpret. • For transparency, please specify whether costs are reported in constant-year USD and include the price year/inflation basis in Table S1 to support reproducibility. Reviewer #2: (No Response) ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: No Reviewer #2: Yes: Sennai Mesfun ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. https://doi.org/10.1371/journal.pone.0343968.r003
Revision 2
9 Feb 2026 Author Response Review Comments to the Author: Reviewer #1: The reply and manuscript’s text adjustments of Reviewer #1 comments were highlighted in yellow. We thank the Reviewer for the thorough review of the manuscript. • Please standardize units/notation (e.g., g/L, kg/kg, US$/kg) and define all acronyms at first mention (AER, SC, HD, CEH/SEH/UCEH) to improve readability. Reply: Thank you for the comment. We have revised the manuscript to standardize all units and notation across the text, tables, and figures. Additionally, all acronyms and abbreviations were revised. • Please add a brief justification (or citation) for the selected enzyme dosages and hydrolysis duration to strengthen comparability with prior studies. Reply: Thank you for the comment. The enzyme dosages and hydrolysis durations were selected based on the technical datasheets and usage recommendations provided by the enzyme supplier (Proenzimas). This information has been included in the Materials and Methods section, together with a reference to the supplier’s technical documentation. • Please clarify the intended syrup product specification (e.g., target solids content and typical sugar profile) so the selling-price assumption is easier to interpret. Reply: Thank you for the comment. We assumed a final composition of the glucose syrup as approximately 90% glucose, 1.2% maltose, and 1.2% maltotriose, with traces of minor soluble and proteins. This information was added to the text. • For transparency, please specify whether costs are reported in constant-year USD and include the price year/inflation basis in Table S1 to support reproducibility. Reply: Thank you for the comment. We have added the following information to Table S1: Reference year: 2025 (all costs expressed in constant 2025 USD. Inflation index: 2025 (the capital cost was adjusted using SuperPro V14 built-in values of the Chemical Engineering Cost Index). Reviewer #2: (No Response) Attachments Attachment Submitted filename: Response_to_Reviewers_auresp_2.docx https://doi.org/10.1371/journal.pone.0343968.r004
15 Feb 2026 Decision Letter - Leonidas Matsakas, Editor Enzymatic hydrolysis of starch from the anthocyanin extraction residue (AER-starch) with ultrasound pretreatment: a techno-economic assessment PONE-D-25-60146R2 Dear Dr. Osorio-Tobón, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support . If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Leonidas Matsakas Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0343968.r005
Formally Accepted
Acceptance Letter - Leonidas Matsakas, Editor PONE-D-25-60146R2 PLOS One Dear Dr. Osorio-Tobón, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Leonidas Matsakas Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0343968.r006

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .