Behavior of Phenolic Compounds During In Vitro Digestion of an Isotonic Beverage Enriched with Microencapsulated Habanero Pepper Leaf Extracts

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “Stability of Phenolic Compounds During In Vitro Digestion of an Isotonic Beverage Enriched with Microencapsulated Habanero Pepper Leaf Extracts” presents a novel and relevant study integrating natural deep eutectic solvent (NADES)-based extraction with microencapsulation technology for valorizing agro-industrial by-products. The work is well-aligned with current trends in sustainable food processing and functional beverage development. The experimental design is thorough, combining cytotoxicity assessment, acute oral toxicity evaluation, and detailed in vitro digestion analyses under both fasting and postprandial conditions.

Overall, the study is original, methodologically sound, and potentially impactful. However, some aspects require clarification, minor corrections, and enhancement to improve clarity and reproducibility.

Major Comments

1. Justification of NADES composition

   • While the selection of choline chloride and glucose is supported by previous work, the rationale for the specific 1:0.8 molar ratio and 68% water should be better contextualized with reference to extraction efficiency and compound stability.

2. Standard food matrix composition

   • The nutritional rationale for the macronutrient proportions is mentioned, but a brief justification for the use of corn-based components over other options would enhance reproducibility and interpretation in the context of Mexican dietary habits.

3. Polyphenol degradation discussion

   • The discussion would benefit from a deeper mechanistic explanation of the observed stability differences between fasting and postprandial conditions, particularly regarding interactions with food macronutrients.

4. Interpretation of antioxidant capacity results

   • The lower antioxidant capacity of the enriched beverage compared to standard food in some digestion stages should be critically discussed—e.g., possible matrix effects, compound transformation, or interference with the DPPH assay.

5. Figures and tables clarity

   • Figures 2–5 contain many abbreviations; a more comprehensive legend or visual simplification would aid non-specialist readers.

Minor Comments

1. Ensure consistency in units (e.g., “mg/100 ml” vs. “mg per 100 ml”).

2. Correct minor typographical errors (e.g., “rxtract” → “extract” in section headings).

3. Provide HPLC chromatograms for key phenolic standards in the supplementary section for transparency.

4. Include the limit of detection (LOD) and limit of quantification (LOQ) for polyphenol analyses.

5. In the abstract, consider adding the % increase in TPC for the enriched beverage compared to the control for clarity.

Author Response

"Please see the attachment."

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Title: Stability of Phenolic Compounds During In vitro Digestion of an Isotonic Beverage Enriched with Microencapsulated Habanero Pepper Leaf Extracts

Manuscript No: Processes3817928

The paper is scientific good but some typographical mistakes are there. Discussion and conclusion should be improved. SOme comments are given below:

Line no 48-49: The manuscript currently uses inconsistent citation styles. Please ensure that only one citation style is followed throughout the text, in accordance with the journal’s guidelines. Avoid mixing different formats (e.g., numbered citations and author–year style).

Line no 89: Avoid using absolute terms like “no studies” as they may not be accurate and can weaken the scientific tone. Instead, use more appropriate phrases such as “limited studies,” “seldom reported,” or “scarce literature” to maintain precision and academic integrity.

Section 2.1: In the "Plant Material" section, you mentioned the use of black soil; however, it is important to also describe the growing conditions in detail. Please include information on soil composition, type and source of irrigation water, and the frequency of irrigation. These agronomic factors can significantly influence the plant’s biochemical properties, including phenolic content and other functional compounds, and are essential for reproducibility and interpretation of results.

Section 2.2: In the “Sorting of Leaves” section, please specify the criteria used for selecting leaves for further analysis. Mention the characteristics or properties considered—such as color, size, maturity, physical integrity, or absence of disease. Clearly explaining the sorting method will enhance the transparency and reproducibility of your experimental process.

Line no 117:  You mentioned that ground leaves were directly used for extraction. Please clarify whether the powdered material was used immediately or stored prior to extraction. If storage was involved, specify the storage conditions (e.g., temperature, duration, container type) to ensure clarity and reproducibility of the methodology, as storage conditions can impact the stability of bioactive compounds.

Line no 121: For all abbreviated terms (e.g., Habanero pepper leaves [HPRE]), please ensure that the full form is mentioned only once at the first occurrence, followed by the abbreviation in parentheses. After that, use only the abbreviation throughout the manuscript. Repeating the full form multiple times disrupts the flow and is not necessary once the abbreviation has been defined.

Line no 129: Please provide specific details regarding the storage of the sample material. Mention the type of storage container used, the temperature and duration of refrigeration, and the purpose of refrigeration (e.g., to preserve phytochemicals, prevent microbial growth, or maintain sample integrity). This information is essential for ensuring reproducibility and understanding how storage conditions may influence the quality and stability of the samples.

Line no 160: OECD. What is this. Required full abbreviation.

Line no 164: Please specify the basic parameters or indicators you monitored to assess illness or abnormal behavior in the subjects or plants (e.g., changes in color, texture, wilting, leaf drop, growth rate, feeding behavior, or signs of infection). Including these observations is important to ensure the health and reliability of the biological material used in the study.

Line no 167: You mentioned maintaining a range of 46% to 59%, but such a wide fluctuation (13%) lacks precision and creates vagueness. If you are claiming that a parameter was "maintained," the range should be much narrower (e.g., ±2–5%) to reflect controlled conditions. Please justify the reason for this variation, or revise the statement to accurately reflect the level of control maintained during the experiment.

Line no 192 and 213: Avoid starting sentences with "To..." as it can lead to incomplete or awkward sentence structures if not properly constructed. While it is grammatically acceptable in some cases (e.g., "To evaluate the effect..."), ensure that such usage forms a complete and clear sentence. Review and revise these instances to improve sentence clarity and flow.

Section 2.3: Please mention the detailed sample composition used for spray drying, including all ingredients, concentrations, and proportions.

Line no 206: Do not use vague terms like "room temperature" in scientific writing. This lacks precision and creates ambiguity. You must mention the exact temperature (e.g., 25 ± 2°C). Such imprecise terminology is unacceptable for reproducible research. Avoid repeating this mistake in future submissions.

Line no 241-242: The formula (𝐴𝑎𝑑𝑗 − 𝐴𝑠𝑝 / 𝐴𝑎𝑑𝑗 ) is unclear and lacks proper formatting and explanation. Please define each variable clearly (e.g., 𝐴𝑎𝑑𝑗, 𝐴𝑠𝑝), and provide a proper mathematical structure for readability. Additionally, include a brief explanation of the formula immediately below it, as commonly done in well-structured scientific articles. Refer to published literature for standard formatting practices and revise accordingly to maintain clarity and professionalism.

Lino no 284-285: What is Fasting period, Please be specific..

Please correct figure 1. There is Line no. 371 and 372 in between the figure.

The color bars in your figure are not clearly labeled, making it difficult to understand which bar corresponds to which sample or concentration. Each color must be explicitly justified and linked to its respective treatment group (e.g., SDS [100 µg/mL], NADES-68 at various concentrations such as 6.3%, 3.1%, 1.6%, 0.78%). Additionally, clarify what the mentioned percentages represent—whether they indicate concentration, proportion of solvent, or another variable. Proper legend annotation and figure explanation are essential for accurate interpretation of your results.

The explanation of results must be grounded in scientific reasoning and should directly align with the procedures described in the Materials and Methods section. Currently, there is a disconnect—key experimental details mentioned in the results are missing from the methods, and there is inadequate scientific explanation in the discussion. Ensure consistency and coherence throughout the manuscript. All findings must be supported by methodological details and discussed with proper scientific rationale.

Line no 383-385: You mention a slight increase in body weight in all individuals, suggesting normal growth and the absence of adverse effects. However, this observation must be supported with a clear scientific explanation. Please provide possible biological or physiological mechanisms behind this weight gain—such as improved metabolic activity, absence of toxicity, enhanced nutrient absorption, or homeostatic regulation. Cite relevant scientific literature to justify the trend. Observational statements without mechanistic support weaken the scientific value of your discussion.

The graph shows a reduction in I-ErL (intestinal early time sampling) for CBP and EBP under postprandial conditions, followed by an increase. Please explain the underlying biological or physiological mechanisms responsible for this trend. Why does this pattern occur specifically with CBP and EBP, and not with other samples or conditions? A scientific rationale is essential. Additionally, support your interpretation by comparing and validating your findings with previously published research to strengthen the credibility of your results.

Line no 653: To improve the clarity and scientific strength of your discussion, begin by explaining the main issues encountered in the study by Bezerra et al. (2025), particularly the reason why they were unable to evaluate cytotoxicity—such as the extremely low pH (<3) of their lactic acid:glucose-based NADES, which may have led to cell instability or interference with assay conditions. Clearly outline the scientific troubleshoot or limitation they faced. Then, contrast those challenges with your own findings, where you successfully evaluated sugar-based NADES extracts. Provide the mechanisms that may explain your successful outcome—such as adjusted pH, use of biocompatible components, or optimized extraction conditions—and support this with relevant literature. This logical flow will make your discussion more coherent, comparative, and scientifically informative.

Line no 665: OECD guidelines. Mention it full form. So, we can understand.

Line no 673-675: This comparison with Bezerra et al. (2025) is not appropriate in the main body of the discussion, as it breaks the flow of your findings. If you wish to include this point, it should be placed either in the last paragraph of the introduction—to highlight the research gap—or in the conclusion, to strengthen the significance of your successful evaluation. Avoid inserting unrelated comparative statements within the core discussion without a clear contextual link.

Line 800-814: You mention Quercetin and its quality indices, but the relevance to your study is unclear. Please clarify how this information is directly linked to your experimental findings. If there is no specific correlation or application within your study framework, this section appears unnecessary. Avoid including generic or widely available information (easily found through general sources like Google) unless it adds scientific value or supports your results. Focus on incorporating relevant, study-specific data that strengthens your discussion.

Conclusion: The conclusion should briefly summarize the key findings of the study without repeating detailed data, highlight the scientific significance and practical implications of those results, and clearly state how the study contributes to existing knowledge. It should also reflect on any limitations, if relevant, and suggest directions for future research or application. Avoid introducing new data or vague statements. Instead, focus on providing a clear, evidence-based closing statement that reinforces the value of your work and aligns with your objectives.

Author Response

"Please see the attachment."

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Stability of Phenolic Compounds During In vitro Digestion of an Isotonic Beverage Enriched with Microencapsulated Habanero Pepper Leaf Extracts

Overall comments:

This study extracted polyphenolic compounds from the leaves of the Habanero pepper (Capsicum chinense Jacq.), a plant originating from the Yucatán Peninsula. Microencapsulation technology was then applied to prevent the degradation of polyphenols in the extract. Subsequently, the product was developed into a beverage and tested for cytotoxicity and acute toxicity to evaluate the biological activity of the polyphenols. The novelty of the study lies in integrating polyphenols extracted using NADES from Habanero pepper leaves into a functional beverage. This research is significant in utilizing agricultural by-products to enhance the value of agricultural products, particularly given that up to 80% of Habanero pepper by-products remain underutilized.

Specific comments:

1. It seems that the title of the article does not fully reflect the research content and objectives. Specifically, the extensive work carried out to evaluate toxicity is not included in the title. In addition, the study also determined the polyphenol profile in the beverage, not just aimed at assessing stability. Furthermore, the stability of phenolic compounds was not evaluated in the research results. Therefore, to better align with the study, the title should be revised.
2. The Introduction and Materials and Methods sections are presented quite well.
3. The Results section should be reorganized for better coherence. The sections on phenol content evaluation (Sections 3.2 and 3.3) should be presented first in the Results before moving on to the toxicity evaluation.
4. The Discussion section should be shortened. Additionally, more illustrative figures, images, or tables should be included to provide evidence and comparisons.

Author Response

"Please see the attachment."

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript addresses a relevant topic: application of agro-industrial byproducts, containing flavonoids, for health benefits. By now, flavonoids from Habanero pepper leaves for enriching isotonic beverage were not produced by ultrasound-assisted extraction with Natural Deep Eutectic Solvents, for this reason investigation of this process and microencapsulation technologies for the purpose of stability under physiological conditions fills important gaps in the presented topic.

Authors provided all necessary information and references for understanding the novelty and significance of presented research. All steps of study are described sufficiently: application of choline chloride/glucose mixture in water for ecologically friendly extraction. The quality of natural material is undoubtable, methods are reliable. Sonication was used to intensify extraction.

Safety of the extract’s at the limit dose 662 of 2000 mg/kg was estimated by cytotoxicity measurements; prepared herbal extract obtained with NADES-68 and ultrasonic probe. Authors concluded that NADES-68 formulation is well tolerated at the tested dose level, sodium dodecyl sulfate was used as positive control.

Encapsulating agents are reported, and microencapsulation process was performed perfectly. Isotonic beverage, prepared from purified water, 6% sugar, some table salt and red fruit flavoring, was enriched with 4% w/v the of the microencapsulated product.

Modifications to previously reported methods for determination of total polyphenols and antioxidant capacity were developed. To simulate the feeding conditions reliable ingredients homogenized in water were applied, such as corn cooking oil, corn flour, granulated sugar, meat peptone and casein peptone. Adjusting pH for modelling was described. Statistical analysis is performed using stat graphics Centurion XVII.II X64 software.

Complex experimental work was carried out to achieve the aim of study. Figures and tables are easy to understand, references are formatted well. Conclusions are supported by results; main ideas of the study are summarized well.

The manuscript can be published after minor revision:

• Ultrasound-assisting is not mentioned in the conclusion. Could you at least highlight in the Introduction part to what extent the yield of extraction enhances during this procedure compared to extraction with NADES only (excluding sonification)?
• Ref 6: 2023 in bold; Refs 10, 11, 17 and 49: 2024 in bold; ref. 15: 2022 in bold; ref. 24: 2021 in bold; ref. 61: 2025 in bold; refs 71, 72: please put year after Journal name.

Author Response

"Please see the attachment."

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Authors had made all corrections, now paper is in good order.