Unlocking the Hidden Microbiome of Food: The Role of Metagenomics in Analyzing Fresh Produce, Poultry, and Meat

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper “Unlocking the Hidden Microbiome of Food: The Role of Metagenomics in Analyzing Fresh Produce, Poultry, and Meat” highlights the importance of applying metagenomics to track microorganisms along the food chain, including their transition from farming environments (farms or pastures) into fresh meat and meat products.

However, the stated objective focuses on showcasing the capabilities and applications of metagenomics in food analysis, including food safety, quality, and microbiome dynamics, without explicitly emphasizing the analysis of the entire production-to-consumption chain.

The analyses presented in the article cover specific aspects such as pathogen detection, microbiome traceability, and the influence of environmental factors on the microbial composition in meat and derived products. Nevertheless, these details are not directly integrated into the formulation of the study’s objective, creating a certain dissonance between the stated aim and the depth of the analyses provided.

The paper loses originality, as similar reviews already exist in the field.

In the conclusions, the statement regarding the lack of bioinformatics expertise, the difficulty of distinguishing living from dead organisms, and the complexity of the food matrix is mentioned as a challenge. However, it is not discussed in detail within the article. It is briefly mentioned without being supported by an in-depth analysis or concrete examples from the study. This gives the impression that it is a generic statement inserted without sufficient grounding in the article's content.

Author Response

We sincerely appreciate the comments and suggestions to improve our manuscript "Unlocking the Hidden Microbiome of Food: The Role of Metagenomics in Analyzing Fresh Produce, Poultry, and Meat." We have incorporated the suggested changes to enhance the discussion on metagenomics and metabolomics, improve data visualization, and include relevant information on food fraud detection. Below, we provide detailed responses to each point raised:

Comment 1: The stated objective does not explicitly emphasize the analysis of the entire production-to-consumption chain.

Response: We appreciate this observation. While the article's primary focus is to highlight the capabilities and applications of metagenomics in food analysis, we have revised the objective to reflect its relevance more explicitly across the entire production-to-consumption chain. As a result, explicit references to how microorganisms migrate along this chain, from agricultural environments to derived meat products, have been incorporated. This adjustment aims to better align the objective with the scope of the analyses presented.

Comment 2: The dissonance between the stated objective and the depth of the analyses provided.

Response: We acknowledge the need to strengthen the connection between the stated objective and the analyses presented. To address this concern, we have included a more detailed summary in the introduction, contextualizing how the aspects analyzed (pathogen detection, microbiome traceability, and the influence of environmental factors) relate to the study's overarching objective. Additionally, we have added a brief discussion at the end of the manuscript that links these areas of analysis to the production-to-consumption chain.

Comment 3: The manuscript lacks originality, as similar reviews exist.

Response: Thank you for this comment. In the discussion section, we have emphasized the unique aspects of this article. Specifically, we highlight comparing specific metagenomic techniques (such as metabarcoding, shotgun sequencing, and functional metagenomics) in the context of different food matrices (fresh produce, meat, and poultry). We have also included recent examples of innovative applications, such as the use of predictive AI in metagenomics, to underscore the originality of our review.

Comment 4 The conclusions mention challenges (lack of bioinformatics expertise, difficulty distinguishing live from dead organisms, complexity of the food matrix), but these are not discussed in depth.

Response: We have expanded the discussion in the limitations section to provide a more in-depth analysis of these challenges. Specific examples have been included, such as implementing host DNA depletion protocols to address the complexity of food matrices and integrating automated bioinformatics pipelines to overcome technical expertise limitations. Additionally, we have presented a discussion of emerging techniques that could facilitate the differentiation between live and dead microorganisms.

Reviewer 2 Report

Comments and Suggestions for Authors

Below are some questions and suggestions that the author should address in the manuscript:

 

1.      The author has primarily focused on metagenomic approaches. However, it would be beneficial to discuss the advantages of metagenomics over metabolomics in the context of food analysis. A comparison between these two approaches, particularly in terms of their capabilities and limitations, would add value to the manuscript.

2.      How can metagenomic techniques be applied to differentiate food fraud (e.g., meat adulteration), especially in cases where microbial toxins are involved? This could be a crucial aspect in improving food safety and authenticity.

3.      The author should ensure they have obtained copyright permission for Figure 1, as this is necessary for proper publication.

4.      It would be helpful to include a statistical graph that shows the number of publications related to the metagenomic analysis of fresh produce, poultry, and meat over the years (from 2000 to 2025). This would provide context and demonstrate the growing interest in this field.

5.      I encourage the author to include more graphs and figures to illustrate key points and findings. This would enhance the manuscript’s clarity and visual appeal.

6.      A table summarizing the various types of food analyzed for freshness using metagenomic approaches would be valuable. This would help to provide a clear overview of the different food groups studied and their microbial characteristics.

Author Response

We sincerely appreciate the comments and suggestions to improve our manuscript "Unlocking the Hidden Microbiome of Food: The Role of Metagenomics in Analyzing Fresh Produce, Poultry, and Meat." We have incorporated the suggested changes to enhance the discussion on metagenomics and metabolomics, improve data visualization, and include relevant information on food fraud detection. Below, we provide detailed responses to each point raised:

Comment 1: The author has primarily focused on metagenomic approaches. However, it would be beneficial to discuss the advantages of metagenomics over metabolomics in the context of food analysis. A comparison between these two approaches, particularly in terms of their capabilities and limitations, would add value to the manuscript.

Response: Thank you for this insightful comment. We have expanded the section "Limitations of Metagenomics" to include a detailed comparison between metagenomic and metabolomic techniques in food analysis. This comparison highlights the unique strengths of metagenomics, such as microorganisms' taxonomic and functional identification. It contrasts these with the strengths of metabolomics, such as the direct detection of toxic chemical compounds and metabolites. Additionally, we address the limitations of both techniques, including the sensitivity of metagenomics to degraded DNA and the limited coverage of metabolites in metabolomics.

Comment 2: How can metagenomic techniques be applied to differentiate food fraud (e.g., meat adulteration), especially in cases where microbial toxins are involved? This could be a crucial aspect in improving food safety and authenticity.

Response: We have included a new subsection in the "Current Applications of Metagenomics in Food Safety" section, addressing metagenomic techniques for detecting food fraud, such as meat adulteration. This discussion explains how metagenomics can identify species-specific DNA of undeclared animal sources, even in complex food matrices. Additionally, we analyze the potential of these techniques to detect microbial toxins associated with food fraud by leveraging functional data derived from shotgun metagenomics and advanced bioinformatics tools such as MetaPhlAn and FAPROTAX.

Comment 3: The author should ensure they have obtained copyright permission for Figure 1, which is necessary for proper publication.

Response: We acknowledge this observation and confirm that copyright permission for the original Figure 1 could not be obtained due to a lack of response from the authors. To address this, we have generated a new figure with updated data showing the evolution of scientific publications on metagenomics in food safety. The figure is titled:

"Number of scientific publications available in the literature on metagenomics for food safety."

This new figure maintains the original intent of providing context on the field's growth while ensuring compliance with editorial requirements.

Comment 4: It would be helpful to include a statistical graph that shows the number of publications related to the metagenomic analysis of fresh produce, poultry, and meat over the years (from 2000 to 2025). This would provide context and demonstrate the growing interest in this field.

Response: We have added a new statistical figure illustrating the number of publications related to metagenomic analysis of fresh produce, poultry, and meat from 2000 to 2024. The data for this figure were extracted from SCOPUS using relevant keywords related to the manuscript's topic. This figure has been incorporated into the introduction to contextualize the growing scientific interest in this field.

Comment 5: A table summarizing the various types of food analyzed for freshness using metagenomic approaches would be valuable. This would provide a clear overview of the food groups studied and their microbial characteristics.

Response: We have included a table summarizing the various types of foods analyzed for freshness using metagenomic approaches. The table details the food types studied (e.g., fresh produce, meat, poultry), the main techniques used (e.g., metabarcoding and shotgun sequencing), and the identified microbial characteristics. This table has been added to the "Metagenomics of Different Types of Edible Products" section to provide a clear and systematic overview of the food groups analyzed.

Reviewer 3 Report

Comments and Suggestions for Authors

I commend the authors for their insights in preparing this manuscript. However, I have a few suggestions for them. 

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,
We are grateful for your valuable feedback on our manuscript "Unlocking the Hidden Microbiome of Food: The Role of Metagenomics in Analyzing Fresh Produce, Poultry, and Meat." Your comments have greatly helped us enhance the content's clarity, coherence, and depth. Below, we address each observation and provide details on the modifications made.

Comment 1: The summary is too general and does not reflect the research findings.

Response: The summary has been restructured to include more specific details about the study's key findings. It now more accurately mentions the applications of metagenomics in pathogen detection, microbiome traceability, and the identification of antimicrobial resistance genes in fresh produce, meat, and poultry. The significant challenges in implementing these technologies in the food industry are also highlighted.

Comment 2: The introduction is too brief and lacks a clear topic transition.

Response: We have expanded the introduction to provide a more solid context before introducing metagenomics. The following points are now explained in greater detail:

• The evolution of food safety, from traditional methods to the need for advanced molecular tools.
• The role of metagenomics in pathogen detection and antimicrobial resistance, emphasizing its advantages over conventional techniques.
• The impact of globalization and climate change on food safety, justifying the relevance of metagenomic studies.

These modifications ensure a more precise and more coherent introduction.

Comment 3: A better justification for why fresh produce harbors large bacterial populations is needed.

Response: A detailed explanation has been added regarding the factors influencing the microbiota of fresh produce, including:

• Physical and chemical factors, such as high water activity and neutral pH.
• Environmental interactions, such as soil, irrigation, and storage, influence microbial diversity.

Additionally, the claim about the relationship between microbiota and allergic diseases has been clarified with updated references.

Comment 4: Transitions between topics are abrupt and hinder readability.

Response: We have reorganized the paragraphs for the poultry and meat sections to improve the narrative flow. Now:

• A more precise transition is provided between microbiome characterization, pathogen detection, and its impact on public health.
• Explicit connections have been added between the studies mentioned to avoid abrupt jumps in the discussion.
• The section on microbial dynamics in poultry farms has been improved, highlighting how these findings can translate into disease control measures.

Comment 5: The challenges section mentions general issues without delving into solutions.

Response: A new section titled "Challenges and Limitations of Metagenomics in Food Safety" has been added, where concrete solutions are now included for:

• The shortage of bioinformatics experts highlights the development of more accessible and automated tools.
• Differentiating between live and dead microorganisms, emphasizing emerging techniques such as PMA-qPCR and metatranscriptomics.
• The complexity of food matrices, proposing the optimization of DNA extraction protocols and selective enrichment techniques.

Comment 6: Some sections deviate into general microbiology without a clear connection to metagenomics.

Response: All sections have been reviewed to ensure each discussion is explicitly linked to metagenomics. For instance, in the antimicrobial resistance section, we emphasize how metagenomic techniques allow for identifying resistance genes and monitoring their spread throughout the food chain.

Comment 7: Minor grammar and writing errors need to be corrected.

Response: A thorough review of the manuscript has been conducted to correct grammatical errors and improve the clarity of the text.

Comment 8: It is suggested that a section on the future of metagenomics in food safety be included.

Response: The manuscript does not include a specific section on future perspectives. However, different parts of the document have mentioned emerging trends, such as the use of long-read technologies and integration with other omics.

Should the editorial committee consider a dedicated future perspectives section necessary, we can work on its inclusion in a revised manuscript.

 

 

Reviewer 4 Report

Comments and Suggestions for Authors

Introduction

Revision: The introduction provides a solid foundation, outlining the importance of food safety and the role of metagenomics in revolutionizing microbial studies.

Critical Points:

1. The first sentence is general; a more specific statistic or example about foodborne illnesses would engage the reader.
2. The challenges mentioned (bioinformatics expertise and distinguishing live vs. dead organisms) could be expanded with examples to highlight their practical implications.

 

Metagenomics of Different Types of Edible Products

Revision: The section is well-organized and categorizes different food types effectively. The explanation of techniques like high-throughput sequencing is clear and informative.

Critical Points:

1. Fresh Produce:
• The section discusses the microbiota well but lacks a detailed analysis of potential mitigation strategies for spoilage and contamination.
• References to studies are relevant but could be synthesized to avoid redundancy.
2. Poultry and Meat:
• Strong discussion on microbial contamination and safety concerns.
• However, the focus on antibiotic resistance could be expanded to include specific regulatory or industrial interventions.
• The role of viral metagenomics in addressing zoonotic diseases is a valuable inclusion but requires further elaboration on its practical applications.

 

Challenges in Metagenomics

Revision: The challenges are articulated well, with a focus on technical and methodological issues.

Critical Points:

1. The section could benefit from a comparative table summarizing the key limitations of current techniques.
2. The discussion on the need for bioinformatics expertise is relevant but would be stronger with examples of recent advances or potential solutions.

 

Conclusion

Revision: The conclusion effectively reiterates the transformative potential of metagenomics in food analysis, emphasizing its implications for food safety and quality.

Critical Points:

1. While the conclusion is comprehensive, it could provide a clearer call-to-action for future research or industry practices.
2. The mention of "widespread adoption" lacks specificity. Highlighting key areas or stakeholders for implementation would make it more actionable.

 

General Comments

 

1. References: The manuscript uses extensive citations, which adds credibility, but some references could be streamlined for relevance.
2. Figures and Tables: Visual aids like comparative tables or charts summarizing microbial diversity findings across different food types would enhance readability.

Author Response

We sincerely appreciate the comments and suggestions to improve our manuscript "Unlocking the Hidden Microbiome of Food: The Role of Metagenomics in Analyzing Fresh Produce, Poultry, and Meat." We have incorporated the suggested changes to enhance the discussion on metagenomics and metabolomics, improve data visualization, and include relevant information on food fraud detection. Below, we provide detailed responses to each point raised:

Comment 1: The first sentence is general; a more specific statistic or example about foodborne illnesses would engage the reader.

Response: We appreciate your comment. We have revised the opening sentence of the introduction to include a relevant statistic about foodborne illnesses. Specifically, we now mention that, according to the World Health Organization, foodborne illnesses affect 600 million people annually, underscoring the critical importance of food safety. This adjustment aims to capture the reader's attention better and establish context from the outset.

Comment 2: The challenges mentioned (bioinformatics expertise and distinguishing life from dead organisms) could be expanded with examples to highlight their practical implications.

Response: We have expanded the discussion of these challenges in the introduction. For example, we describe how the lack of bioinformatics expertise can limit the interpretation of metagenomic data in the food industry and how the inability to differentiate between live and dead organisms affects microbiological risk assessments. The practical examples mentioned in the "Limitations of Metagenomics" section support these expansions.

Comment 3: Fresh Produce: The section discusses the microbiota well but lacks a detailed analysis of potential mitigation strategies for spoilage and contamination.

Response: We have added a more detailed analysis of potential strategies to mitigate spoilage and contamination in fresh produce. This includes postharvest management practices such as using microbiome-based biocontrol agents and advanced cold storage methods that intentionally modify microbial communities to extend shelf life.

Comment 4: References to studies are relevant but could be synthesized to avoid redundancy.

Response: We have revised the section to synthesize the references, removing repetitions and grouping related studies. This improves the flow of the text and reduces redundancy without compromising the depth of the discussion.

Comment 5: Poultry and Meat: The focus on antibiotic resistance could be expanded to include specific regulatory or industrial interventions.

Response: We have expanded the discussion on antibiotic resistance in poultry and meat to include examples of regulatory and industrial interventions, such as implementing "antibiotic-free production" programs and using alternatives like prebiotics and probiotics in animal feed.

Comment 6: The role of viral metagenomics in addressing zoonotic diseases is a valuable inclusion but requires further elaboration on its practical applications.

Response: We have further developed the discussion on viral metagenomics by including practical applications, such as identifying emerging viruses in livestock and their impact on public health. This is complemented by recent case studies demonstrating how viral metagenomics can inform mitigation strategies for zoonotic diseases.

Comment 7: The section could benefit from a comparative table summarizing the key limitations of current techniques.

Response: We have added a comparative table to this section summarizing the main limitations of current metagenomic techniques (e.g., shotgun sequencing, metabarcoding, and long-read sequencing) along with proposed solutions. This table facilitates a clearer understanding of the challenges and areas for improvement.

Comment 8: The discussion of the need for bioinformatics expertise is relevant, but it would be more assertive with examples of recent advances or potential solutions.

Response: We have included examples of recent advances, such as automated bioinformatics tools and more accessible analysis pipelines (e.g., QIIME2 and MetaPhlAn). Additionally, we highlight training initiatives in bioinformatics to bridge the expertise gap in the food industry.

Comment 9:  The conclusion effectively reiterates the transformative potential of metagenomics in food analysis, emphasizing its implications for food safety and quality.

Response: We have adjusted the conclusion to include a more explicit call to action, emphasizing the need for collaboration among researchers, regulators, and industry to implement metagenomic standards in food safety. Key areas for adoption, such as the standardization of analysis protocols and the development of accessible bioinformatics infrastructure, are also identified.

Comment 10: References: The manuscript uses extensive citations, which adds credibility, but some references could be streamlined for relevance.

Response: We have reviewed the references and removed those not essential to the manuscript's main points. This improves conciseness and enhances the relevance of the citations.

Comment 11:Figures and Tables: Visual aids like comparative tables or charts summarizing microbial diversity findings across different food types would enhance readability.

Response: Additional figures and tables, such as comparative charts summarizing microbial diversity findings across various food types and a visual workflow of metagenomic processes, have been included. These additions improve the manuscript's clarity and visual appeal.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Author responded all raised question