Natural Antimicrobials from Plants Used as Food Preservatives

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript addresses a highly relevant and timely topic, namely the use of plant-derived antimicrobial compounds as natural alternatives to synthetic preservatives in food systems. Given the increasing demand for clean-label products and sustainable preservation strategies, this topic is of clear importance for the food science community. The manuscript covers a broad range of literature and provides a structured overview of major phytochemical classes, their mechanisms of action, and potential applications. That being said, although the manuscript has a solid foundation, it currently reads more as a descriptive compilation rather than a critical review. The main shortcomings relate to limited data synthesis, insufficient critical discussion, and a weak connection between in vitro findings and real food applications. In its present form, the manuscript would benefit from substantial revision in order to improve both scientific depth and practical relevance.

Abstract:

• The abstract is clearly organized and reflects the overall scope of the manuscript. However, it remains rather general and does not fully convey the key insights emerging from the review. At present, it lacks quantitative or comparative elements that would strengthen its scientific impact. It would be beneficial to include more specific statements regarding the effectiveness of different compound classes, as well as the main challenges associated with their use in food systems. Adding even a single representative example of antimicrobial activity could significantly improve clarity and relevance.

1. Introduction:

• The introduction provides a solid background on food safety challenges, including microbial contamination, foodborne diseases, and food waste. The inclusion of statistical data contributes to the relevance of the discussion, and the transition toward natural alternatives is logical and well justified. At the same time, certain parts of the section could be streamlined, as some ideas are repeated. The discussion of synthetic preservatives would also benefit from a more balanced perspective, taking into account regulatory limits and safe usage levels. In addition, a clearer distinction between spoilage microorganisms and pathogenic bacteria would improve scientific precision and readability.

2. Main classes and sources of plant-based antimicrobials:

• This section demonstrates extensive literature coverage and is generally well structured. The classification of compounds is appropriate, and the manuscript successfully summarizes their sources and key characteristics. However, the section tends to remain descriptive. For example, while essential oils and terpenoids are well presented, their practical limitations such as volatility, instability, and strong sensory impact are not sufficiently discussed. Similarly, the section on polyphenols is well organized and highlights relevant factors such as lipophilicity, but it would benefit from a clearer comparison with other compound classes. The subsections on alkaloids, sulfur-containing compounds, and saponins often follow a listing approach. These parts would be strengthened by a more critical perspective, particularly regarding toxicity, bioavailability, and feasibility of application in food systems. Including brief integrative paragraphs summarizing the advantages and limitations of each group would greatly improve this section.

 

• Table 1: Table 1 compiles a large amount of data and is clearly the result of thorough literature research. Nevertheless, its current format makes interpretation difficult. The use of different parameters (MIC, inhibition zones, CPSI, LD50), often expressed in different units, complicates direct comparison. To enhance clarity, it would be advisable to standardize the data or separate different assay types into distinct tables. Grouping the results by compound class and including a qualitative indication of antimicrobial strength could further improve usability.

3. Modes of antimicrobial action of plant-derived compounds:

• This is one of the strongest parts of the manuscript. The mechanisms are described in detail and supported by relevant literature, covering membrane disruption, inhibition of nucleic acid and protein synthesis, and metabolic interference. Despite the solid scientific basis, the section would benefit from better integration. The mechanisms are presented somewhat independently, without sufficient discussion of their overlap or potential synergistic effects. A short concluding paragraph emphasizing the multi-target nature of phytochemicals would enhance the overall coherence. In addition, while resistance is briefly mentioned, this aspect deserves more attention, especially considering its relevance for long-term application.

4. Antimicrobial compounds applications in the food industry:

• This section is particularly important given the scope of the manuscript, yet it appears somewhat underdeveloped. Although several examples are provided, the discussion does not fully address the challenges of translating in vitro activity into real food systems.The role of the food matrix especially interactions with proteins, lipids, and other components is not sufficiently considered, even though it is known to significantly affect antimicrobial performance. Organizing the section according to food categories could improve clarity and allow for more meaningful comparisons. In addition, sensory aspects and technological solutions, such as encapsulation or nanoemulsions, should be discussed in greater detail to strengthen the practical relevance of this section.

 

• Table 2: Table 2 provides useful insights into applications in food systems, but its current presentation limits comparability. The outcomes are not consistently reported, which makes it difficult to draw clear conclusions. Standardizing parameters such as microbial reduction or shelf-life extension, along with clearly indicating the food matrix and concentrations used, would improve the table considerably. A short paragraph summarizing the main trends observed would also be beneficial.

5. Challenges and safety considerations:

• This section is essential but currently underdeveloped. Important aspects such as toxicity, acceptable intake levels, and regulatory frameworks (EFSA, FDA) are only briefly mentioned. Similarly, the discussion of stability is rather limited and should be expanded to include environmental and matrix-related factors. A clearer structure separating technological challenges from safety and regulatory aspects would improve both readability and scientific depth.

6. Conclusion:

• The conclusion provides a general summary of the topic but remains somewhat broad. It would be strengthened by a more focused synthesis of the main findings, including identification of the most promising compound classes and key limitations. A more forward-looking perspective, highlighting future research directions and practical implementation strategies, would further enhance its impact.

Author Response

The manuscript addresses a highly relevant and timely topic, namely the use of plant-derived antimicrobial compounds as natural alternatives to synthetic preservatives in food systems. Given the increasing demand for clean-label products and sustainable preservation strategies, this topic is of clear importance for the food science community. The manuscript covers a broad range of literature and provides a structured overview of major phytochemical classes, their mechanisms of action, and potential applications. That being said, although the manuscript has a solid foundation, it currently reads more as a descriptive compilation rather than a critical review. The main shortcomings relate to limited data synthesis, insufficient critical discussion, and a weak connection between in vitro findings and real food applications. In its present form, the manuscript would benefit from substantial revision in order to improve both scientific depth and practical relevance.

Answer: We sincerely thank the reviewer for recognizing the relevance and timeliness of our manuscript, as well as the structured overview provided on major phytochemical classes, their mechanisms of action, and potential applications. We also appreciate the constructive feedback regarding areas for improvement. In response to the concerns raised, we have undertaken a substantial revision of the manuscript to enhance its scientific depth and practical relevance. We hope that these revisions have transformed the manuscript from a descriptive compilation into a critical review that provides both scientific insight and practical guidance for the use of plant-derived antimicrobials in food preservation.

Abstract. The abstract is clearly organized and reflects the overall scope of the manuscript. However, it remains rather general and does not fully convey the key insights emerging from the review. At present, it lacks quantitative or comparative elements that would strengthen its scientific impact. It would be beneficial to include more specific statements regarding the effectiveness of different compound classes, as well as the main challenges associated with their use in food systems. Adding even a single representative example of antimicrobial activity could significantly improve clarity and relevance.

Answer: We thank the reviewer for this constructive feedback. We agree that the original abstract, while organized, could better convey the key insights and specific findings of the review. In response, we have revised the abstract to include comparative statements regarding the effectiveness of different phytochemical classes, highlight representative examples of antimicrobial activity, and clearly summarize the main challenges associated with their application in food systems. These changes aim to enhance the clarity, relevance, and scientific impact of the abstract. Below, highlighted in blue, you can see the corrected version as it now appears in the text:

“Plant-derived antimicrobial compounds are emerging as promising alternatives to synthetic preservatives in the food industry due to their efficacy against a broad spectrum of pathogenic and spoilage microorganisms, as well as their consumer acceptance. This review critically examines the main classes of bioactive phytochemicals, including essential oils, polyphenols, alkaloids, terpenoids, and saponins, comparing their relative antimicrobial effectiveness and highlighting representative examples. Notably, essential oils rich in thymol or carvacrol have shown strong inhibitory activity against Listeria monocytogenes and Salmonella spp., while polyphenols and alkaloids exhibit moderate to strong activity depending on concentration and food matrix. Their mechanisms of action include cell membrane disruption, inhibition of key enzymes, and interference with DNA or protein synthesis. Applications in food systems (i.e., incorporation into coatings, emulsions, or controlled-release formulations) demonstrate potential for extending shelf life and enhancing safety. However, practical implementation is challenged by matrix-dependent efficacy, compound stability, sensory impact, and regulatory and toxicological considerations. By synthesizing current knowledge, identifying the most promising compound classes, and highlighting key limitations, this review provides a critical framework to guide future research and the development of effective, sustainable natural preservatives in the food industry.”

Introduction. The introduction provides a solid background on food safety challenges, including microbial contamination, foodborne diseases, and food waste. The inclusion of statistical data contributes to the relevance of the discussion, and the transition toward natural alternatives is logical and well justified. At the same time, certain parts of the section could be streamlined, as some ideas are repeated. The discussion of synthetic preservatives would also benefit from a more balanced perspective, taking into account regulatory limits and safe usage levels. In addition, a clearer distinction between spoilage microorganisms and pathogenic bacteria would improve scientific precision and readability.

Answer: We thank the reviewer for this detailed and constructive feedback. We have revised the introduction to improve clarity, streamline repetitive content, and distinguish more clearly between spoilage microorganisms and pathogenic bacteria. Additionally, we have moderated the discussion of synthetic preservatives to emphasize their efficacy and regulatory safety, while acknowledging potential risks under high-exposure or experimental conditions. These changes aim to improve readability, scientific accuracy, and the balance of the discussion.

Main classes and sources of plant-based antimicrobials. This section demonstrates extensive literature coverage and is generally well structured. The classification of compounds is appropriate, and the manuscript successfully summarizes their sources and key characteristics. However, the section tends to remain descriptive. For example, while essential oils and terpenoids are well presented, their practical limitations such as volatility, instability, and strong sensory impact are not sufficiently discussed. Similarly, the section on polyphenols is well organized and highlights relevant factors such as lipophilicity, but it would benefit from a clearer comparison with other compound classes. The subsections on alkaloids, sulfur-containing compounds, and saponins often follow a listing approach. These parts would be strengthened by a more critical perspective, particularly regarding toxicity, bioavailability, and feasibility of application in food systems. Including brief integrative paragraphs summarizing the advantages and limitations of each group would greatly improve this section.

Answer: We thank the reviewer for their constructive feedback on the section “Main classes and sources of plant-based antimicrobials.” In response, the text has been revised to provide a more critical and comparative perspective for each compound class. The revisions highlight class-specific characteristics such as stability, potency, bioavailability, and regulatory considerations, and include comparative remarks among essential oils, polyphenols, alkaloids, sulfur-containing compounds, terpenoids, and saponins to inform practical applicability in food systems. Integrative paragraphs summarizing the advantages and limitations of each class have been added, while avoiding repetition of general limitations (e.g., sensory impact, solubility) that are already discussed in the “Challenges and Safety Considerations” section. These changes improve the section’s critical depth and cohesion with the rest of the manuscript, strengthening both its scientific insight and practical relevance.

Table 1. Table 1 compiles a large amount of data and is clearly the result of thorough literature research. Nevertheless, its current format makes interpretation difficult. The use of different parameters (MIC, inhibition zones, CPSI, LD50), often expressed in different units, complicates direct comparison. To enhance clarity, it would be advisable to standardize the data or separate different assay types into distinct tables. Grouping the results by compound class and including a qualitative indication of antimicrobial strength could further improve usability.

Answer: We thank the reviewer for their careful evaluation of Table 1. We fully agree that the heterogeneity of parameters (MIC, inhibition zones, CPSI, LD50) and the variability of units can complicate direct comparison—a common issue in antimicrobial literature reviews, and one that is frequently encountered even when comparing results from prior experimental studies. To mitigate this, we have standardized all units that were originally reported inconsistently (e.g., µg/mL converted to mg/mL) to facilitate comparability.

Regarding the suggestion to divide the table into multiple smaller tables, we considered this option but concluded that it would substantially increase the number of tables, resulting in some that would be too short to provide meaningful context. Instead, we incorporated the assay type directly in the results column, and we classified all entries according to compound class. Each compound class is indicated by a horizontal row acting as a header for that group. Additionally, when extract characterization was performed, the specific compounds identified are included in parentheses in the “Extraction” column. This structure allows readers to contextualize the data efficiently while maintaining a single, comprehensive table.

Concerning the suggestion of including a qualitative indication of antimicrobial strength, we decided not to incorporate this. Assigning qualitative ratings would necessarily involve subjective judgments about what constitutes “strong” or “weak” activity, which may vary depending on the assay conditions and the specific microbial target. We therefore opted to present the numerical and experimental data directly, allowing readers to interpret antimicrobial potency based on standardized units and reported results.

Modes of antimicrobial action of plant-derived compounds. This is one of the strongest parts of the manuscript. The mechanisms are described in detail and supported by relevant literature, covering membrane disruption, inhibition of nucleic acid and protein synthesis, and metabolic interference. Despite the solid scientific basis, the section would benefit from better integration. The mechanisms are presented somewhat independently, without sufficient discussion of their overlap or potential synergistic effects. A short concluding paragraph emphasizing the multi-target nature of phytochemicals would enhance the overall coherence. In addition, while resistance is briefly mentioned, this aspect deserves more attention, especially considering its relevance for long-term application.

Answer: We thank the reviewer for recognizing the detailed presentation of antimicrobial mechanisms. We agree that discussing the overlap and potential synergistic effects of these mechanisms can enhance the section's coherence. To address this, we have added a short concluding paragraph highlighting the multi-target nature of plant-derived compounds, emphasizing how simultaneous disruption of membranes, interference with nucleic acid and protein synthesis, and metabolic perturbation can contribute to enhanced antimicrobial efficacy and reduce the likelihood of resistance development.

Regarding the reviewer’s comment on resistance, we have expanded the discussion to more explicitly address this issue, including the potential for sub-inhibitory concentrations to induce adaptive responses in microorganisms and the implications for long-term use in food systems. This addition aims to provide a more critical perspective on the practical challenges and limitations associated with prolonged application of phytochemicals, while maintaining a focus on their multi-target modes of action.

Below, highlighted in blue, you can see the corrected version as it now appears in the text:

“In summary, phytochemicals exert antimicrobial activity through a diverse array of mechanisms that collectively compromise microbial viability. These multi-target effects act synergistically, simultaneously compromising multiple cellular processes, which enhances overall antimicrobial efficacy and limits the ability of microorganisms to develop stable resistance [150]. At the same time, sub-inhibitory exposures can induce transient stress responses, such as upregulation of efflux pumps or other adaptive pathways, particularly under prolonged or repeated use. This highlights that, despite their multi-target nature, careful attention to formulation, dosing, and delivery strategies is required to maintain effective concentrations and prevent tolerance development [151]. By understanding the molecular targets and interactions of phytochemicals, it will be possible not only to rationally design novel antimicrobial formulations but also to strategically combine compounds to maximize synergistic effects while minimizing the risk of resistance. These features underscore the promise of plant-derived compounds as versatile alternatives or complements to conventional antimicrobials in both food preservation and therapeutic contexts.”

Antimicrobial compounds applications in the food industry. This section is particularly important given the scope of the manuscript, yet it appears somewhat underdeveloped. Although several examples are provided, the discussion does not fully address the challenges of translating in vitro activity into real food systems. The role of the food matrix especially interactions with proteins, lipids, and other components is not sufficiently considered, even though it is known to significantly affect antimicrobial performance. Organizing the section according to food categories could improve clarity and allow for more meaningful comparisons. In addition, sensory aspects and technological solutions, such as encapsulation or nanoemulsions, should be discussed in greater detail to strengthen the practical relevance of this section.

Answer: We thank the reviewer for this valuable and constructive comment. We would like to clarify that the section was already organized according to major food categories (meat products, horticultural products, bakery products, and packaging materials), which we believe provides a clear framework for application-oriented discussion.

In response to the reviewer’s suggestions, we have revised the section to strengthen its analytical depth and practical relevance. Greater emphasis has been placed on the challenges associated with translating in vitro antimicrobial activity into real food systems, particularly highlighting the role of the food matrix and its interactions with proteins, lipids, and other components that may reduce efficacy.

In addition, we have expanded the discussion within each subsection to better reflect application-specific constraints and variability among food categories. Sensory aspects and technological strategies, such as encapsulation and nanoemulsion-based delivery systems, have been incorporated in a concise and contextualized manner. Care has been taken to avoid redundancy with the “Challenges and Safety Considerations” section by focusing here on their practical implications within each food system, while maintaining a more detailed and general discussion of these limitations in the dedicated section.

Table 2. Table 2 provides useful insights into applications in food systems, but its current presentation limits comparability. The outcomes are not consistently reported, which makes it difficult to draw clear conclusions. Standardizing parameters such as microbial reduction or shelf-life extension, along with clearly indicating the food matrix and concentrations used, would improve the table considerably. A short paragraph summarizing the main trends observed would also be beneficial.

Answer: We thank the reviewer for this valuable comment regarding Table 2. We agree that improving comparability and clarity would enhance its usefulness. In response, we have revised the table to ensure a more consistent presentation of outcomes. Where possible, the reported effects have been standardized to include comparable parameters such as microbial reduction (e.g., log CFU reduction) or shelf-life extension. We have also clarified key information, including the food matrix and the concentrations of antimicrobial compounds used, to facilitate interpretation and comparison across studies. In addition, we have included a brief summary paragraph in the manuscript highlighting the main trends observed across the different applications. This paragraph emphasizes the variability in efficacy depending on the food matrix, the generally stronger performance observed in simplified systems compared to complex foods, and the relevance of formulation strategies in improving antimicrobial effectiveness.

Challenges and safety considerations. This section is essential but currently underdeveloped. Important aspects such as toxicity, acceptable intake levels, and regulatory frameworks (EFSA, FDA) are only briefly mentioned. Similarly, the discussion of stability is rather limited and should be expanded to include environmental and matrix-related factors. A clearer structure separating technological challenges from safety and regulatory aspects would improve both readability and scientific depth.

Answer: We thank the reviewer for highlighting the importance of strengthening the “Challenges and Safety Considerations” section. We agree that this part of the manuscript required further development to fully address key aspects related to practical implementation. In response, we have substantially revised this section to improve both its structure and scientific depth. The content has been reorganized to clearly distinguish between technological challenges and safety and regulatory considerations, enhancing readability and coherence. The discussion of technological aspects has been expanded to include stability issues under different environmental conditions and the influence of the food matrix on compound performance. Additionally, the section now provides a more detailed overview of safety considerations, including toxicity, acceptable intake levels, and current regulatory frameworks, with specific reference to authorities such as EFSA and FDA. These additions aim to offer a more comprehensive and critical perspective on the factors limiting the industrial application of plant-derived antimicrobials.

Overall, these revisions strengthen the section by providing a more structured, balanced, and in-depth analysis of the key challenges associated with the use of these compounds in food systems.

Conclusion. The conclusion provides a general summary of the topic but remains somewhat broad. It would be strengthened by a more focused synthesis of the main findings, including identification of the most promising compound classes and key limitations. A more forward-looking perspective, highlighting future research directions and practical implementation strategies, would further enhance its impact.

Answer: We thank the reviewer for this constructive suggestion. We have revised the conclusion to provide a more focused synthesis of the main findings, highlighting the compound classes with the most promising antimicrobial activity and the key challenges that remain. Additionally, we have included a forward-looking perspective, emphasizing future research directions and practical strategies for the implementation of plant-derived antimicrobials in the food industry, while maintaining a balanced and evidence-based tone. Below, highlighted in blue, you can see the corrected version as it now appears in the text:

“Phytochemicals isolated from plants represent a potential alternative to synthetic additives in the food industry. Among these, essential oils, phenolic compounds, alkaloids, terpenes, and saponins have shown notable activity against foodborne pathogens. However, although plant-derived antimicrobials hold significant potential as environmentally friendly alternatives to synthetic preservatives, their practical application in the food industry still faces several key challenges, including standardization, matrix-dependent efficacy, toxicological evaluation, and regulatory approval. Addressing these challenges will be critical to developing effective natural preservatives that maintain microbiological safety while preserving the organoleptic and nutritional quality of foods. Future research should aim to deepen our understanding of the mechanisms of action of individual phytochemicals and their interactions within com-plex food matrices. Advances in formulation science, such as encapsulation, con-trolled-release systems, and combination strategies, can enhance stability, efficacy, and sensory compatibility. Additionally, integrating predictive approaches like metabolomics and computational modeling can help anticipate interactions and optimize ap-plications. Alongside thorough toxicological and regulatory evaluation, these strategies will facilitate the practical implementation of plant-derived antimicrobials in commercial foods, supporting more sustainable and safe preservation solutions while opening avenues for innovative, application-oriented research in the field.”

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

Your manuscript addresses a highly relevant and timely topic, namely, the potential use of plant-derived natural antimicrobials as alternatives to synthetic food preservatives. I found the topic highly relevant and appropriate for the journal. The manuscript covers a broad and interesting area; however, in its current form, I feel that it remains somewhat descriptive and would benefit from major revision.

• Lines 66–68: I would suggest softening the statement regarding the role of synthetic additives in the selection of resistant bacteria. As currently phrased, it sounds too categorical for a review article and would benefit from a more cautious formulation.
• Lines 89–94: The scope of the review is clear, but I think the manuscript would be stronger if the authors stated more explicitly what is new here compared with previous reviews on natural preservatives and plant-derived antimicrobials.
• Lines 101–103: I would recommend revising the statement that the long history of plant use facilitates regulation and food application. In my view, traditional use does not automatically address present toxicological or regulatory concerns, especially when concentrated extracts or isolated compounds are involved.
• Lines 121–122: This sentence should be rewritten for grammar and clarity. At present, the wording is awkward and interrupts the flow of the section.
• More generally, I felt that the section on phytochemical classes reads at times as a list of examples. I would encourage the authors to synthesize the literature more clearly and indicate which classes are most promising, which are best supported by evidence, and which still face substantial formulation or stability limitations.
• The mechanistic section is useful and generally well organized. That said, I think the manuscript should more clearly distinguish between mechanisms that are well established and those that are still proposed or inferred indirectly.
• Lines 455–470: In my opinion, the discussion of synthetic preservatives needs a more balanced tone. Some of the wording may overemphasize potential risks without clearly separating experimental/high-exposure findings from exposure under approved food-use conditions.
• Lines 478–490: I would recommend moderating the language in this paragraph. Terms such as “remarkable efficacy” and the suggestion that these compounds are likely to gain broad commercial presence seem somewhat stronger than the later discussion of limitations would support.
• One of the main issues, in my view, is that the manuscript does not always clearly separate in vitro or model-system results from evidence obtained in real food matrices. This distinction is essential and should be maintained more consistently throughout the review.
• Lines 631–665: I found the discussion of active packaging, extract variability, matrix effects, and compound instability particularly important. In fact, for me, this is one of the strongest parts of the paper. For that reason, I think these limitations should be brought more explicitly into the earlier sections, rather than appearing mainly toward the end.
• Lines 728–775: The regulatory discussion is very relevant, and the conclusion should better reflect it. Personally, I would tone down phrases such as “have proven to be a promising alternative” and especially “paradigm shift,” because the manuscript itself makes clear that major challenges remain regarding standardization, matrix-dependent efficacy, toxicological evaluation, and regulatory approval.

Comments on the Quality of English Language

The manuscript is generally understandable, but the English could be improved in several places. For example, there is awkward phrasing in Lines 121–122 , overly strong wording in Lines 61–68 , and a rather exaggerated tone in the conclusion (Lines 761–775)

Author Response

Dear authors,

Your manuscript addresses a highly relevant and timely topic, namely, the potential use of plant-derived natural antimicrobials as alternatives to synthetic food preservatives. I found the topic highly relevant and appropriate for the journal. The manuscript covers a broad and interesting area; however, in its current form, I feel that it remains somewhat descriptive and would benefit from major revision.

Answer: We sincerely thank the reviewer for the positive evaluation of our work and for recognizing the relevance and timeliness of the topic addressed in our manuscript. We appreciate the constructive feedback regarding the overall structure and depth of the paper. In response to this comment, we have undertaken a thorough revision of the manuscript to reduce its descriptive nature and enhance its analytical depth. We have also refined several sections to improve coherence and ensure a more focused and critical perspective throughout the manuscript. We believe that these revisions have significantly improved the quality and scientific rigor of the paper.

Lines 66–68: I would suggest softening the statement regarding the role of synthetic additives in the selection of resistant bacteria. As currently phrased, it sounds too categorical for a review article and would benefit from a more cautious formulation.

Answer: We thank the reviewer for this valuable suggestion. We agree that the original wording may have been too categorical for a review article. Following this comment, we have revised the sentence to adopt a more cautious and balanced tone, better reflecting the current state of the evidence and the inherent complexity of this issue. All changes made to the manuscript were carried out using the "Track and Changes" tool. Below, in blue, you can see the corrected version that now appears in the text:

“This is because some studies suggest that the intensive use of these synthetic additives may contribute to the selection of resistant bacteria in both industrial and natural environments.”

Lines 89–94: The scope of the review is clear, but I think the manuscript would be stronger if the authors stated more explicitly what is new here compared with previous reviews on natural preservatives and plant-derived antimicrobials.

Answer: We thank the reviewer for this insightful comment. We agree that explicitly highlighting the novelty of our review strengthens the manuscript and helps better position it within the existing literature. In response, we have revised this section to clearly state the distinctive contribution of our work compared to previous reviews. Below, in blue, you can see the corrected version that now appears in the text:

“In this context, this review provides a comprehensive overview of plant-derived anti-microbial compounds, including their principal classes and natural sources, mechanisms of action, and applications in food systems. Beyond summarizing existing knowledge, the manuscript adopts an integrated approach that connects these aspects with current challenges and safety considerations. Particular emphasis is placed on bridging fundamental insights with practical applications, offering an updated and more application-oriented perspective on the use of plant-derived antimicrobials in food preservation.”

Lines 101–103: I would recommend revising the statement that the long history of plant use facilitates regulation and food application. In my view, traditional use does not automatically address present toxicological or regulatory concerns, especially when concentrated extracts or isolated compounds are involved.

Answer: We thank the reviewer for this thoughtful and important comment. We fully agree that traditional use does not necessarily guarantee safety under current conditions, particularly when dealing with concentrated extracts or isolated compounds. In response, we have revised the statement to avoid overgeneralization and to better reflect the need for proper toxicological evaluation and regulatory assessment. The revised text now presents a more cautious and accurate perspective. Below, highlighted in blue, you can see the corrected version as it now appears in the text:

“While this extraction can be carried out from various biological sources, most studies focus on the plant kingdom, as plants represent an accessible and cost-effective matrix with a long history of use. However, despite this background, the safety, regulatory approval, and application of concentrated extracts or isolated compounds still require thorough evaluation before their incorporation into food systems”.

Lines 121–122: This sentence should be rewritten for grammar and clarity. At present, the wording is awkward and interrupts the flow of the section.

Answer: We thank the reviewer for pointing this out. We agree that the original sentence could be improved in terms of clarity and flow. Accordingly, we have revised the text to enhance readability and ensure a smoother transition between ideas within the section. Below, highlighted in blue, you can see the corrected version as it now appears in the text:

“In this context, investigating the role of individual EO components may provide a deeper understanding of these mechanisms and reveal effects that remain obscured when EOs are studied as complex mixtures. This knowledge could ultimately support the development of more targeted and effective applications in the food industry, as highlighted by numerous studies reporting the antimicrobial properties of EOs derived from a wide range of plant tissues…”

More generally, I felt that the section on phytochemical classes reads at times as a list of examples. I would encourage the authors to synthesize the literature more clearly and indicate which classes are most promising, which are best supported by evidence, and which still face substantial formulation or stability limitations.

Answer: We thank the reviewer for their constructive feedback on the section “Main classes and sources of plant-based antimicrobials.” The revised text now presents a more critical and comparative perspective for each compound class, emphasizing key characteristics such as stability, potency, bioavailability, and regulatory considerations. Essential oils and terpenoids are discussed in terms of both their strong antimicrobial activity and practical limitations, including volatility, instability, and sensory impact, while polyphenols are compared with other classes to highlight differences in lipophilicity, efficacy, and matrix interactions. Similarly, alkaloids, sulfur-containing compounds, and saponins are analyzed not only for their potential antimicrobial properties but also for the challenges they pose regarding toxicity, bioavailability, and feasibility of application in food systems. Integrative paragraphs summarize the advantages and limitations of each class, providing a clear and balanced evaluation that supports informed decisions on their practical use. Overall, these revisions enhance the critical depth, comparative clarity, and practical relevance of the section.

The mechanistic section is useful and generally well organized. That said, I think the manuscript should more clearly distinguish between mechanisms that are well established and those that are still proposed or inferred indirectly.

Answer: We thank the reviewer for this observation. In response, we have revised the mechanistic section to more clearly distinguish between mechanisms that are well established and those that are proposed or inferred indirectly. Established mechanisms, such as membrane disruption, inhibition of nucleic acid and protein synthesis, and metabolic interference, are supported by direct experimental evidence, whereas other effects, including interference with quorum sensing, biofilm modulation, or synergistic multi-target interactions, are discussed as emerging or inferred based on indirect observations. This distinction is now explicitly indicated in the text, allowing readers to differentiate between strongly supported mechanisms and those that remain hypothetical or context-dependent. By providing this clarification, the section maintains its scientific rigor while highlighting areas where further experimental validation is warranted.

Lines 455–470: In my opinion, the discussion of synthetic preservatives needs a more balanced tone. Some of the wording may overemphasize potential risks without clearly separating experimental/high-exposure findings from exposure under approved food-use conditions.

Answer: We thank the reviewer for this important observation. We agree that some sections of the original text could overemphasize potential risks of synthetic preservatives without clearly distinguishing between high-exposure experimental conditions and typical use levels. We have revised the paragraph to present a more balanced and nuanced discussion, highlighting both the efficacy and established safety of these additives under approved food-use conditions. Below, highlighted in blue, you can see the corrected version as it now appears in the text:

“Regarding propionates, these additives are particularly effective against mold growth and are widely used in industrial baking to prevent rancidity and mold in doughs and breads. Regulations and scientific reviews generally classify them as safe when used according to good manufacturing practices, although some literature investigates acute effects on human metabolism [148]. Some experimental studies under high-exposure conditions have reported transient effects on glucose metabolism or hormonal responses; however, such effects have not been observed at typical consumption levels [149]. Sulfites and sulfating agents, including sulfur dioxide, sodium and potassium sulfite, metabisulfite, and bisulfites, are used in canned goods and, above all, in wine production to inhibit malolactic fermentation [150]. While certain individuals may experience sensitivity reactions, such as headaches or bronchoconstriction in asthmatic patients, these effects are generally associated with higher exposures than those occurring under standard food-use conditions [151–153]. Similarly, antifungal agents such as natamycin (or pimaricin) are applied to the surface of cheeses and some sausages to prevent mold and yeast growth. Due to its low solubility, natamycin remains primarily on the surface, and studies indicate a favorable safety profile when used in accordance with authorized food-use regulations [154]. Collectively, these findings highlight that, while potential risks have been identified under experimental or high-exposure conditions, synthetic preservatives remain effective and safe when used as intended in food systems.”

Lines 478–490: I would recommend moderating the language in this paragraph. Terms such as “remarkable efficacy” and the suggestion that these compounds are likely to gain broad commercial presence seem somewhat stronger than the later discussion of limitations would support.

Answer: We thank the reviewer for this suggestion. We agree that some of the original wording was stronger than the evidence presented and could overstate the potential of these compounds. We have revised the paragraph to adopt a more balanced and cautious tone, aligning the description of efficacy with the limitations discussed later in the manuscript. Below, highlighted in blue, you can see the corrected version as it now appears in the text:

“These compounds, including essential oil constituents, polyphenols, terpenoids, and organic acids, have shown significant activity against a range of pathogenic and spoilage microorganisms through distinct mechanisms of action, making them promising candidates for food preservation [155]. Additionally, many of these natural compounds exhibit complementary functional properties, such as antioxidant capacity, which can help limit deterioration processes like lipid oxidation in meat or browning in fruit [156,157]. Advances in formulation science and food technology are contributing to improved efficacy, stability, and applicability of these natural preservative systems, suggesting that their use in commercial foods may increase in the future. This approach offers a potential strategy to support food safety, quality, and sustainability, while acknowledging the challenges that remain in their practical implementation.”

One of the main issues, in my view, is that the manuscript does not always clearly separate in vitro or model-system results from evidence obtained in real food matrices. This distinction is essential and should be maintained more consistently throughout the review.

Answer: We thank the reviewer for this important observation. We have carefully revised the manuscript to more clearly distinguish between in vitro or model-system studies and those conducted in actual food matrices. In particular, we have revised the sections on antimicrobial activity, food applications, and the challenges of natural preservatives to specify when results are derived from laboratory assays versus real food systems. For example, we now highlight how factors such as matrix composition, water activity, pH, and interactions with proteins or lipids can significantly modulate antimicrobial efficacy in foods compared with in vitro conditions. Additionally, when discussing specific compound classes and their applications in meat, bakery, horticultural products, and packaging materials, we explicitly indicate whether the reported effects were observed in controlled laboratory conditions or under practical food-processing and storage scenarios. These changes ensure that readers can clearly interpret the relevance and limitations of each study, improving the scientific rigor and practical applicability of the review.

Lines 631–665: I found the discussion of active packaging, extract variability, matrix effects, and compound instability particularly important. In fact, for me, this is one of the strongest parts of the paper. For that reason, I think these limitations should be brought more explicitly into the earlier sections, rather than appearing mainly toward the end.

Answer: We thank the reviewer for highlighting the discussion on active packaging, extract variability, matrix effects, and compound instability as one of the strengths of the manuscript. In response to this suggestion, we have revised the earlier sections of the manuscript (particularly the sections on main classes and sources of plant-derived antimicrobials and applications in food systems) to incorporate these limitations more explicitly. For example, when describing each compound class, we now briefly note the potential variability in extract composition, stability issues under processing and storage conditions, and the influence of the food matrix on antimicrobial efficacy, without repeating the more detailed discussion reserved for the Challenges and Safety Considerations section. These changes aims to ensure that readers are aware of these critical limitations from the outset, while maintaining coherence and avoiding redundancy.

Lines 728–775: The regulatory discussion is very relevant, and the conclusion should better reflect it. Personally, I would tone down phrases such as “have proven to be a promising alternative” and especially “paradigm shift,” because the manuscript itself makes clear that major challenges remain regarding standardization, matrix-dependent efficacy, toxicological evaluation, and regulatory approval.

Answer: We thank the reviewer for this constructive feedback. We agree that the original conclusion contained overly strong expressions that could overstate the current state of plant-derived antimicrobials. In response, we have revised the text to adopt a more measured tone, emphasizing the regulatory, safety, and technical challenges that remain, while highlighting the potential of these compounds in a balanced way. Below, highlighted in blue, you can see the corrected version as it now appears in the text:

“Phytochemicals isolated from plants represent a potential alternative to synthetic additives in the food industry. Among these, essential oils, phenolic compounds, alkaloids, terpenes, and saponins have shown notable activity against foodborne pathogens. However, although plant-derived antimicrobials hold significant potential as environmentally friendly alternatives to synthetic preservatives, their practical application in the food industry still faces several key challenges, including standardization, matrix-dependent efficacy, toxicological evaluation, and regulatory approval. Addressing these challenges will be critical to developing effective natural preservatives that maintain microbiological safety while preserving the organoleptic and nutritional quality of foods. Future research should aim to deepen our understanding of the mechanisms of action of individual phytochemicals and their interactions within com-plex food matrices. Advances in formulation science, such as encapsulation, controlled-release systems, and combination strategies, can enhance stability, efficacy, and sensory compatibility. Additionally, integrating predictive approaches like metabolomics and computational modeling can help anticipate interactions and optimize ap-plications. Alongside thorough toxicological and regulatory evaluation, these strategies will facilitate the practical implementation of plant-derived antimicrobials in commercial foods, supporting more sustainable and safe preservation solutions while opening avenues for innovative, application-oriented research in the field.”

The manuscript is generally understandable, but the English could be improved in several places. For example, there is awkward phrasing in Lines 121–122, overly strong wording in Lines 61–68, and a rather exaggerated tone in the conclusion (Lines 761–775).

Answer: We thank the reviewer for the careful reading and constructive feedback regarding the language and tone of the manuscript. We have carefully revised the text throughout the manuscript to improve clarity, readability, and precision.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

The revised manuscript shows clear improvement compared to the previous version. The structure is more coherent, the discussion is more balanced, and the inclusion of practical aspects related to food systems, as well as limitations such as matrix effects, stability, and safety, has strengthened the overall quality of the review. The manuscript now reads as a more critical and application-oriented contribution.

A few points should still be addressed:

• Table 1 has been improved, and the definitions of parameters (MIC, inhibition zones, CPSI) are clearly provided. However, the use of different assay types within the same table still makes direct comparison between results difficult for the reader. It would be helpful to further guide interpretation, for example by more clearly distinguishing assay types, slightly refining the grouping of entries, or adding a brief explanatory note in the caption to clarify how the data should be compared.
• Formatting issues (visible editing marks and inconsistent formatting) should be carefully corrected throughout the manuscript to ensure a clean and professional final version.
• In some parts of the manuscript, particularly within the section describing different classes of compounds, the text still occasionally becomes descriptive, with a tendency to list examples rather than synthesize them. Adding a few brief comparative or integrative sentences would improve the flow and help maintain a consistently critical tone.

Overall, the manuscript is now in good shape and only requires minor revisions.

 

Author Response

Dear Authors,

The revised manuscript shows clear improvement compared to the previous version. The structure is more coherent, the discussion is more balanced, and the inclusion of practical aspects related to food systems, as well as limitations such as matrix effects, stability, and safety, has strengthened the overall quality of the review. The manuscript now reads as a more critical and application-oriented contribution.

A few points should still be addressed:

Table 1 has been improved, and the definitions of parameters (MIC, inhibition zones, CPSI) are clearly provided. However, the use of different assay types within the same table still makes direct comparison between results difficult for the reader. It would be helpful to further guide interpretation, for example by more clearly distinguishing assay types, slightly refining the grouping of entries, or adding a brief explanatory note in the caption to clarify how the data should be compared.

Answer: Thank you very much for your careful assessment and for your positive comments on the improvements made to the manuscript. We agree that the inclusion of different assay types within the same table may make direct comparison less straightforward. To address this, we have revised Table 1 by adding a new column specifying the type of antimicrobial assay used (e.g., MIC/MBC, inhibition zone, CPSI), in order to clearly distinguish between methodologies.

In addition, we have included a brief explanatory note in the text to clarify that results obtained using different assay types are not directly comparable and should be interpreted with caution, particularly when comparing quantitative (MIC) and qualitative (inhibition zone) data.

We believe these modifications improve the clarity of the table and better guide the reader in interpreting the reported results.

Formatting issues (visible editing marks and inconsistent formatting) should be carefully corrected throughout the manuscript to ensure a clean and professional final version.

Answer: Thank you for pointing this out. We agree on the importance of ensuring a clear and professional presentation of the manuscript. The entire document has been carefully revised to remove all visible editing marks and to correct formatting inconsistencies. We have standardized the formatting throughout the manuscript, including text, tables, and references, to ensure a clean and consistent final version.

In some parts of the manuscript, particularly within the section describing different classes of compounds, the text still occasionally becomes descriptive, with a tendency to list examples rather than synthesize them. Adding a few brief comparative or integrative sentences would improve the flow and help maintain a consistently critical tone.

Answer: Thank you for this insightful comment. We have revised these sections to reduce the listing of individual examples and to incorporate more comparative and integrative statements. Specifically, we have added brief sentences that highlight common trends, differences in antimicrobial performance, and recurring limitations across compound classes, in order to strengthen the critical perspective and improve the overall flow of the manuscript. We believe these changes contribute to a more cohesive and analytically robust discussion.

Overall, the manuscript is now in good shape and only requires minor revisions.

Answer: Thank you for your positive evaluation of the revised manuscript. We are pleased to know that you consider it to be in good shape. We have carefully addressed the remaining minor revisions suggested and believe that these final adjustments have further improved the clarity and overall quality of the manuscript. Thank you again for your time and constructive feedback throughout the review process.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed my previous comments and have substantially improved the manuscript. The revised version demonstrates a clearer scope, a more balanced and critical discussion, and a more cautious interpretation of the available evidence. In particular, the authors have moderated overly strong claims, improved the treatment of safety and regulatory aspects, and strengthened the analytical depth of the review.

In my opinion, the revised manuscript now meets the standards required for publication. While the submitted file may still contain some technical formatting issues or visible tracked changes, these do not affect the scientific quality of the work and can be resolved during the editorial process.

I therefore suggest the acceptance of the manuscript for publication.

Author Response

Thank you very much for your thorough evaluation of our revised manuscript. We sincerely appreciate your positive feedback and your recognition of the improvements made in response to your previous comments. We are glad that the revisions have helped to clarify the scope, strengthen the discussion, and provide a more balanced and cautious interpretation of the evidence. Your suggestions were extremely valuable in improving the overall quality of the work. We also take note of the minor technical and formatting issues mentioned and will ensure that they are fully addressed during the editorial process. Thank you again for your time and constructive input.