Antagonistic Activity of Bacteriocin-like Inhibitory Substances from Enterococcus lactis Isolated from the Surface of Jalapeno Pepper against Foodborne Pathogens

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Abstract:

The abstract provides a comprehensive overview of the study's focus on evaluating the antagonistic activity of LAB strains isolated from jalapeno peppers against various foodborne pathogens. 

The abstract lacks mention of the significance or broader implications of the findings, which could provide readers with a clearer understanding of the study's importance.

It could be more concise by avoiding redundant phrases or unnecessary details.

Introduction:

The introduction effectively contextualizes the study within the broader field of LAB research and food safety concerns related to foodborne pathogens. However, it might benefit from highlighting the novelty or gap in knowledge that this study aims to address.

Materials and Methods:

• The methodology section is detailed and well-structured, providing clear steps for experimental procedures. However, it could be enhanced by including more specific information about the criteria used for strain selection and the rationale behind certain experimental decisions.

• The author should be Specify the exact media composition and growth conditions used for culturing LAB strains isolated from jalapeno peppers. Providing this information will ensure reproducibility and allow other researchers to validate the findings.

• The author should be Describe in detail the protocol followed for 16S rRNA sequencing analysis, including the primers used, PCR conditions, and bioinformatics tools employed for sequence analysis and identification.

• The author should be specify modifications or optimizations were made to standard protocols, such as DNA extraction or sequencing procedures, provide a rationale for these changes and detail the steps followed.

Discussion:

• The discussion could also provide more insight into the practical applications of the findings for food safety and industry, including potential challenges or considerations for implementation.

• The author should be provide a comprehensive comparison of the findings with existing literature on LAB isolated from other food sources. 

• The author should be highlight similarities and differences in microbial composition, bacteriocin production, or other relevant factors to contextualize the research within the broader scientific context.

General comments 

• The author should be Ensure consistency in terminology and formatting throughout the manuscript. Use standardized terminology for describing experimental procedures, microbial taxa, and other technical terms to facilitate clarity and understanding.

• The author should be Verify the accuracy of all citations and references cited in the manuscript. Ensure that the citation style adheres to the guidelines specified by the target journal.

• Addressing these technical points will improve the overall quality and rigor of the manuscript, making it more compelling and impactful to readers and reviewers in the field.

Conclusion:

The conclusion provides a succinct summary of the study's key findings and implications. However, it could be strengthened by suggesting specific avenues for further investigation or application of the findings in practical settings.

Comments on the Quality of English Language

Moderate editing of English language required

Author Response

R: Dear reviewer, thank you for all your comments to help us improve our manuscript. Changes addressing your suggestions and concerns were highlighted with the track-changes function of the word processor and are in the main text.

Abstract

The abstract provides a comprehensive overview of the study's focus on evaluating the antagonistic activity of LAB strains isolated from jalapeno peppers against various foodborne pathogens.

1. The abstract lacks mention of the significance or broader implications of the findings, which could provide readers with a clearer understanding of the study's importance. It could be more concise by avoiding redundant phrases or unnecessary details.

R: Thank you for your observation, the abstract was modified, and the significance of the study was added at the end as follows: “LAB isolated from the surface of jalapeno pepper produce HR-BLIS (possibly enterocin) that exhibited a broad-spectrum antagonistic activity against foodborne pathogens; therefore, are a promising source of natural antimicrobials to ensure food safety”

Introduction

2. The introduction effectively contextualizes the study within the broader field of LAB research and food safety concerns related to foodborne pathogens. However, it might benefit from highlighting the novelty or gap in knowledge that this study aims to address.

R: Thank you for your observation, lines 52-54 were modified in the introduction section of the manuscript to provide a better background. Highlighting the lack of effectiveness of bacteriocins against Gram-negative bacteria.

Materials and Methods

3. The methodology section is detailed and well-structured, providing clear steps for experimental procedures. However, it could be enhanced by including more specific information about the criteria used for strain selection and the rationale behind certain experimental decisions.

R: Thank you for your suggestion, the section was reviewed to make sure all criteria were added to clarify the methods.

4. The author should specify the exact media composition and growth conditions used for culturing LAB strains isolated from jalapeno peppers. Providing this information will ensure reproducibility and allow other researchers to validate the findings.

R: Thank you for your observation, additional information of growth conditions is now mentioned in lines 83-86 as follows: “LAB strains were cryopreserved at -80 °C and were reactivated by inoculation in Man, Rogosa & Sharpe (MRS) broth (DIFCO, USA) pH 7.0 ± 0.2 at 37 °C for 18 h”. By including the media supplier, media composition can be easily verified for anyone that wants to reproduce the experiment.

5. The author should describe in detail the protocol followed for 16S rRNA sequencing analysis, including the primers used, PCR conditions, and bioinformatics tools employed for sequence analysis and identification.
6. The author should specify modifications or optimizations were made to standard protocols, such as DNA extraction or sequencing procedures, provide a rationale for these changes and detail the steps followed.

R: Thank you for your observations, modifications of section 2.5 “LAB 16S rRNA sequence analysis and identification” are now indicated in lines 139-143 Universal primers 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1301R (5’-TACTAGCGATTCCGACTTC-3’) were used. PCR conditions were: initial denaturation at 95 °C for 10 min; 30 denaturation cycles at 95 °C for 1 min each; primer alignment at 55 °C for 1 min and primer extension at 72 °C for 2 min, and a final extension step at 72 °C for 10 min.

Discussion:

7. The discussion could also provide more insight into the practical applications of the findings for food safety and industry, including potential challenges or considerations for implementation.

R: Thank you for your observation, Line 296 were modified as follows “Thermostability is an important and desirable property for new food biopreservatives intended to be used in the food industry, especially if they are going to be used as part of a hurdle system where a thermal processing is commonly required”

8. The author should provide a comprehensive comparison of the findings with existing literature on LAB isolated from other food sources.

R: Thank you for your observation. We considered that in lines 300-303 we are comparing our results (HR-BLIS from E. lactis isolated from jalapeño pepper) with other vegetable sources of LABs which can also produce heat resistant enterocins.  Also, in lines 304-307 we are also comparing our results with published MICs for nisin against the same L. monocytogenes used in our study. Additionally, in lines 316-318 we stated that the effect of HR-BLIS-67 was expected since most bacteriocins are effective against gram (+) bacteria, regardless of their sources. As well in lines 321-327 we mentioned that the antagonistic effect found for HR-BLIS-67 was less effective than those reported for CFS from different types of LABs isolated from cantaloupe, molasses, and cucumber. We agree that we concentrate the comparisons of our results only to other vegetable sources of LABs and not for other food sources such as dairy or meat products.

9. The author should highlight similarities and differences in microbial composition, bacteriocin production, or other relevant factors to contextualize the research within the broader scientific context.

R: Thank you, the manuscript was reviewed. Modification in the discussion section were in lines 327-332 as follows: Differences found between HR-BLIS-67 and these previous reports regarding their ability to inhibit Gram-negative bacteria may be due to differences of several environmental fac-tors which may impact their native microbiome [4]. As a response to harsh environmental factors LABs can generate different antagonistic compounds, including BLIS, which can be more bioactive when compared with the BLIS obtained in the present study from E. lactis.

General comments

10. The author should ensure consistency in terminology and formatting throughout the manuscript. Use standardized terminology for describing experimental procedures, microbial taxa, and other technical terms to facilitate clarity and understanding.

R: Thank you, manuscript was checked, and changes were done.

11. The author should verify the accuracy of all citations and references cited in the manuscript. Ensure that the citation style adheres to the guidelines specified by the target journal.

R: Thank you, all references were checked.

 

Conclusion

12. The conclusion provides a succinct summary of the study's key findings and implications. However, it could be strengthened by suggesting specific avenues for further investigation or application of the findings in practical settings.

R: Thank you, the conclusion was modified as follows: “Enterococcus lactis strain 67 isolated from the surface of jalapeno peppers from Sonora, Mexico possess the ability to produce HR-BLIS (possibly enterocin) that exhibit a broad-spectrum antagonistic activity against L. monocytogenes, S. aureus, S. Typhimurium, and E. coli O157:H7. Therefore HR-BLIS-67, is a promising natural antimicrobial alternative for the control of foodborne pathogens and promote food safety. Further analysis to purify and characterize HR-BLIS-67 should be performed to elucidate its stability and mechanism of action as well as to perform in-situ assays to confirm that its bioactivity is maintained once is used in a food matrix such as meat.”

Reviewer 2 Report

Comments and Suggestions for Authors

The article is dedicated to the search for bacteria producing bacteriocins among lactic acid bacteria. The antimicrobial activity of the supernatants was extensively studied using a wide range of microbiological methods. Intriguing and promising results were obtained, as bacteriocins resistant to 121 degrees Celsius and capable of suppressing gram-positive bacteria are rare and hold significant scientific and industrial interest.
There are some minor queries regarding the methodologies:
1. Why was lyophilization used in preparing the supernatants? Please explain.
2. When using the spot-on-lawn test, it is better to use sterile filter paper discs. These discs can be used independently to prevent false-positive results from washing bacteria off the surface of the agar plate.
Overall, the article is of high scientific quality and recommended for publication in a journal.

Author Response

Dear reviewer, thank you for your comments and suggestions which were very helpful to improve our manuscript. Modifications to the main text were indicated by using the track-changes function of the word processor, therefore, they are clearly visible.

 

The article is dedicated to the search for bacteria producing bacteriocins among lactic acid bacteria. The antimicrobial activity of the supernatants was extensively studied using a wide range of microbiological methods. Intriguing and promising results were obtained, as bacteriocins resistant to 121 degrees Celsius and capable of suppressing gram-positive bacteria are rare and hold significant scientific and industrial interest.

There are some minor queries regarding the methodologies:

1. Why was lyophilization used in preparing the supernatants? Please explain.

R: Thank you for your observation, section 2.2 was modified as follows: “Cell free supernatants of selected LAB were obtained from fresh overnight (18 h growth) cultures, centrifuged (10,000 x g, 10 min, 4 °C), frozen at -80 °C, and freeze-dried (Labconco Freezone 4.5, USA) in order to concentrate the bioactive compounds in the supernatants.”

2. When using the spot-on-lawn test, it is better to use sterile filter paper discs. These discs can be used independently to prevent false-positive results from washing bacteria off the surface of the agar plate.

R: Thank you for your suggestion, we will consider using sterile filter paper discs in further experiments

Reviewer 3 Report

Comments and Suggestions for Authors

In the present study, Hernandez-Mendoza et al. investigated the antibacterial activity of cell-free supernatants from numerous lactic acid bacteria, shedding light on their potential as antimicrobial agents. The authors identified a considerable number of strains, totaling 390, and observed inhibitory effects against Listeria monocytogenes in 72 of these strains. Among the latter, 60 were found to produce bacteriocin-like inhibitory substances, with 16 exhibiting continued antibacterial activity even after exposure to heat treatment. While the article presents several strengths, there are notable areas for improvement.

Major concerns:

A good aspect of the study is the extensive screening of lactic acid bacteria, resulting in the identification of a substantial number of strains with inhibitory effects against L. monocytogenes. However, the authors are not providing figures to support their claims regarding the antibacterial activity of these strains. The authors' claims about the antibacterial activity without quantitative data or visual representations may leave readers questioning the strength of the observations. Integration of figures illustrating the zones of inhibition, accompanied by measurements (if possible) thereof, would strengthen the authors' claims and facilitate a more nuanced interpretation of the results. 

The study's methodology for selecting strains for further analysis is not clear to me, particularly regarding the limited number of strains (4 only) subjected to minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. The rationale behind choosing these particular strains remains unclear. Additionally, the evaluation of antibacterial activity against other bacterial strains is limited to only one strain, which is insufficient for drawing comprehensive conclusions. 

Minor concerns:

Minor concerns include discrepancies observed in starting optical density (OD) values in Figure 2. 

The results depicted in Table 4 need a more comprehensive exploration of the underlying rationale and a thorough explanation of the data presented in the table.

In summary, while the study offers valuable insights into the antibacterial potential of lactic acid bacteria, improvements in data presentation, methodology justification, and discussion clarity would enhance the overall scientific quality and impact of the article.

Author Response

Dear reviewer, thank you for your comments and suggestions that were very helpful to improve our manuscript. Modifications to the main text were indicated by using the track-changes function of the word processor, therefore, they are clearly visible.

In the present study, Hernandez-Mendoza et al. investigated the antibacterial activity of cell-free supernatants from numerous lactic acid bacteria, shedding light on their potential as antimicrobial agents. The authors identified a considerable number of strains, totaling 390, and observed inhibitory effects against Listeria monocytogenes in 72 of these strains. Among the latter, 60 were found to produce bacteriocin-like inhibitory substances, with 16 exhibiting continued antibacterial activity even after exposure to heat treatment. While the article presents several strengths, there are notable areas for improvement.

Major concerns

1. A good aspect of the study is the extensive screening of lactic acid bacteria, resulting in the identification of a substantial number of strains with inhibitory effects against monocytogenes. However, the authors are not providing figures to support their claims regarding the antibacterial activity of these strains. The authors' claims about the antibacterial activity without quantitative data or visual representations may leave readers questioning the strength of the observations. Integration of figures illustrating the zones of inhibition, accompanied by measurements (if possible) thereof, would strengthen the authors' claims and facilitate a more nuanced interpretation of the results.

R: Thank you for your observation, a new Figure 1 (Line 195-197) was included in the manuscript.