Biosafe Control of Staphylococcal Enterotoxins Production in Shelf-Stable Bacon^†

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors:

The manuscript describes the potential utilization of a biopreservative to improve safety against S. aureus.

The manuscript is well written a material and methods are extremely detailed with several data that are not related to the main objective of the work. Thus, headings such as “2.2. Biopreservative production” and “2.3. production of samples” must be largely resumed. Basically, authors must describe the physical-chemical characteristic of each batch since variations in pH, aW and technological treatment (drying, brining or thermal processing) may influence the behavior (i.e. growth) of S. aureus. Also, information about color analysis must be removed since it is not related with the objective of the work.

Regarding the monitorization of the Lag phase and the information indicated in table 3 has no sense. The objective is to verify the growth at specific days and a specific temperature. Table 1 must be indicated in the material and methods section a data about characteristics must be statistically studied. What is the objective of compare 3 food processing techniques if no organoleptical characteristics were evaluated? In contrast, no study about the best food processing technique to control S. aureus was performed. Comparison of B1-T, B2-T and B3-T must be indicated.

However, the main problem of the work is the “biopreservative” used. This does not indicate what kind of biopreservative is used. Is it specific bacteriocin? Is it L. paracasei alone? In the last case, what were the L. paracasei concentration?

Regarding the enterotoxin production, 3 food processing treatments of bacon displayed similar results of SE production. Does differences were detected in contaminated batches w/o treatments? Same question must be clarified for batches treated with the biopreservative.

Regarding the effect of the biopreservative on SE production, it is not discussed properly. It is impossible to discuss what the effect of the biopreservative is when authors did not provide information about it. Even the biopreservative is a patent, the way that the biopreservative interacts or modifies the metabolism related to the toxins formation must be described. In addition, potential synergisms with aW of pH must be assessed.

Overall, the manuscript is well written, but material and methods section indicated several unnecessary pieces of information that did not influence the main objective of the work. Also, the main objective of the work is not correctly studied (i.e. which was the best treatment for S. aureus control? Does biopreservative action vary according to other factors such as aW, pH ot Tº?). Furthermore, the lack of information about the type of biopreservative and how it works prevents the realization of a correct discussion.

Based on the review previously described, the manuscript is not suitable for publication as presented. Since I recognize the value of the work, I suggest authors rewrite the entire manuscript and submit it as a new research article.

Author Response

Comment 1: The manuscript describes the potential utilization of a biopreservative to improve safety against S. aureus.

Response 1: We thank the reviewer for handling our manuscript. Based on your valuable comments, we have substantially improved the quality of the paper, and we hope that the revised version will meet your expectations.

Comment 2:The manuscript is well written a material and methods are extremely detailed with several data that are not related to the main objective of the work. Thus, headings such as “2.2. Biopreservative production” and “2.3. production of samples” must be largely resumed. Basically, authors must describe the physical-chemical characteristic of each batch since variations in pH, aW and technological treatment (drying, brining or thermal processing) may influence the behavior (i.e. growth) of S. aureus. Also, information about color analysis must be removed since it is not related with the objective of the work.

Response 2: We agree with this relevant point raised by the reviewer. The superfluous sections have been removed, and the Materials and Methods were revised accordingly to focus only on information directly related to the objectives of the study.

Comment 3: Regarding the monitorization of the Lag phase and the information indicated in table 3 has no sense. The objective is to verify the growth at specific days and a specific temperature. Table 1 must be indicated in the material and methods section a data about characteristics must be statistically studied.

Response 3: We apologize for the mistake. The information previously presented in Table 3 has been corrected and is now divided into Tables 3A and 3B, in accordance with Reviewer 2’s comments. Additionally, details regarding the Aw of each group have been included. Table 1 is now properly referenced in the Materials and Methods section 2.4, and the statistical analysis has been revised accordingly.

Comment 4: What is the objective of compare 3 food processing techniques if no organoleptical characteristics were evaluated? In contrast, no study about the best food processing technique to control S. aureus was performed. Comparison of B1-T, B2-T and B3-T must be indicated.

Response 4: The SDA Ordinance No. 748 establishes a critical Aw threshold of ≤ 0.85 for shelf-stable bacon. This regulatory limit is based on the growth dynamics of S. aureus and is intended to prevent the SE formation, thereby ensuring the microbiological safety of bacon during non-refrigerated storage. This study highlights the complexity of S. aureus control in shelf-stable bacon, where contamination risks persist even after effective thermal inactivation. While vegetative cells of S. aureus are eliminated during cooking, recontamination during post-lethality handling remains a significant hazard, particularly in environments where moisture accumulates. Crucially, the findings demonstrate that SE production is not solely dependent on the global Aw of the product. Even when Aw values are near 0.85, SE formation can still at the product–package interface. To mitigate this risk, we propose an indirect application of a biopreservative at the product–package interface, thereby providing an additional protective barrier and enhancing product safety. This rationale has been incorporated into the objectives of the study (lines 93-95).

Comment 5: However, the main problem of the work is the “biopreservative” used. This does not indicate what kind of biopreservative is used. Is it specific bacteriocin? Is it L. paracasei alone? In the last case, what were the L. paracasei concentration?

Response 5: Thank you for your consideration. We have added information in lines 584-587 regarding the nature of the preservative.

Comment 6: Regarding the enterotoxin production, 3 food processing treatments of bacon displayed similar results of SE production. Does differences were detected in contaminated batches w/o treatments? Same question must be clarified for batches treated with the biopreservative.

Response 6: In this study, we produced three batches of bacon with different Aw levels. The groups were subjected to one of three treatments: (T3) superficial application of 1.0% biopreservative (treatment), (T2) superficial application of 1.0% sterile distilled water (control), or no superficial application (T1). SE production was assessed by rinsing the surface of the bacon. As clarified in lines 232-233, all groups were superficially contaminated with S. aureus. The results demonstrated that SE production was absent only in the treatment group containing the biopreservative.

Comment 7: Regarding the effect of the biopreservative on SE production, it is not discussed properly. It is impossible to discuss what the effect of the biopreservative is when authors did not provide information about it. Even the biopreservative is a patent, the way that the biopreservative interacts or modifies the metabolism related to the toxins formation must be described. In addition, potential synergisms with aW of pH must be assessed.

Response 7: Thank you for your comment. We have added information in lines 515-523  clarifying that the acidification at the product–package interface constitutes the primary hurdle underlying the antimicrobial efficacy of the biopreservative. The localized decrease in pH generates an unfavorable microenvironment for the growth of S. aureus and consequently inhibits SE formation.

Comment 8: Overall, the manuscript is well written, but material and methods section indicated several unnecessary pieces of information that did not influence the main objective of the work. Also, the main objective of the work is not correctly studied (i.e. which was the best treatment for S. aureus control? Does biopreservative action vary according to other factors such as aW, pH ot Tº?). Furthermore, the lack of information about the type of biopreservative and how it works prevents the realization of a correct discussion. Based on the review previously described, the manuscript is not suitable for publication as presented. Since I recognize the value of the work, I suggest authors rewrite the entire manuscript and submit it as a new research article.

Response 8: We sincerely thank you for handling our manuscript. We carefully considered your valuable comments and have prepared a revised version that we believe represents a substantial improvement. We kindly hope this version will meet your consideration.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The study is generally well structured and presents a valid option for controlling the growth of S. aureus and the production of enterotoxins in shelf-stable bacon according to the principles of hurdle technology. The methodology employed requires clarification at some specific points. The presentation of the results and their discussion need to be improved.

 

Specific comments

Title: Object of biosafe control are not enterotoxins but their production; the title should be modified accordingly: Biosafe control of staphylococcal enterotoxin production …

Abstract: The sentence "Microbial growth and SE production were evaluated under simulated storage using the MicroLab_ShelfLife protocol and ELISA" should be modified: SE production was tested by ELISA. Data about microbial growth and SE production were evaluated under simulated storage using the MicroLab_ShelfLife protocol (or similar).

Keywords: I suggest to add staphylococcal enterotoxins and Lacticaseibacillus paracasei and replace intoxication with foodborne disease.

Introduction

Line 40: Replace “sporous” with “spores”.

Line 44: The achronym SE is explained here but it appears for the first time in Abstract.

Line 45: Please, specify the D reference temperature for enterotoxin A D=28 minutes and correct the typo =of. Other typos are present in the text.

Line 65: Add the verb in the sentence.

Lines 76-78: Please, explain in the scope what type of biostrategy was used.

Materials and methods

2.1: Since a control group not inoculated with S. aureus was not included, were coagulase-positive staphylococci counts performed in the bacon samples before inoculation with S. aureus to verify their absence/quantify their presence?

Lines 88-91: The sentence is not clear. What was the reference for challenge test execution? Add a reference, e.g. https://doi.org/10.4315/0362-028x-73.1.140.

Reformulate the sentence: Then, results from the challenge test were used to evaluate….by the microbial growth predictor…

Add here a reference for MicroLab_ShelfLife.

2.2 Lines 129-131: I suggest to explain here the main characteristics of the produced biopreservative: it is represented by the whole thermally inactivated brothculture of Lacticaseibacillus paracasei DTA 83. Explain here also how it is then prepared before the inoculation in food starting from the 20 L polypropylene containers.

In the Discussion section the authors have to comment how the biopreservative works: bacteriocins?  Organic acids and other active substances, …thermal stability at 80°C…

Even if the strain has been the object of publications cited as references, the text must be self-explanatory in its fundamental points.

2.3 Lines 166-167: S. aureus strain ATCC 25923 is a classical type strain but not the one usually employed for SE production studies, where typical enterotoxigenic ATCC strains such as   ATCC 13565 or ATCC 14458 are used. Please, specify in the text what are the genotypic/phenotypic pattern of SE production of ATCC 25923, with pertinent references.

2.4 Line 201: It seems difficult to get manually 0.3 mm pieces with a blade, maybe 0.3 cm?

Line 229: What does means (T1)?

2.5: Why the minimal bactericidal concentration (MBC) was not determined as well? It would have been very helpful in more accurately interpreting the results of the study.

Line 269: p<0.05, to be consistent with Table 1 and line 370.

2.6 Lines 283-284: Again, not clear (see comment to lines 88-91).

Line 285: English to be revised.

Line 288: 7°C?

Line 291: Why did the authors choose to determine a total bacterial count (ISO 4833) and not a coagulase positive staphylococci count? Please, justify your choice.

Figure 1: A and B: Why is the temperature 5°C and not 7°C?

Results: Table 1: The Aw values were 0.86 (not 0.85), 0.94 (not 0.93) and 0.97 (not 0.95). Please, consider it in the discussion.

Lines 370-371: Clarify this sentence.

Lines 401-402: MBC could have clarified this aspect.

Table 3: I suggest dividing more clearly the table into two parts (A and B) since the first part presents experimental data and the second part predictive data obtained on the basis of experimental data. Modify the Table caption accordingly.

Table 3: Insert an explanation to clarify the lack of data in the Durability row of B1-T and B2-T (e.g. ∞).

Discussion: As anticipated, the specific action of the biopreservative has to be discussed in the framework of the application of the hurdle technology in this specific product and compared with pertinent references about the use of mesophilic lactobacilli cultures or their derivatives vs. S. aureus in bacon or similar products.

Author Response

Comment 1: The study is generally well structured and presents a valid option for controlling the growth of S. aureus and the production of enterotoxins in shelf-stable bacon according to the principles of hurdle technology. The methodology employed requires clarification at some specific points. The presentation of the results and their discussion need to be improved.

Response 1: We are sincerely grateful for your careful handling of our manuscript and for granting us the opportunity to submit a revised version. We have thoroughly addressed each of your comments point by point, and we respectfully hope that this improved version will receive your kind consideration.

Specific comments

Comment 2: Title: Object of biosafe control are not enterotoxins but their production; the title should be modified accordingly: Biosafe control of staphylococcal enterotoxin production …

Response 2: We appreciate your suggestion and have revised the title accordingly.

Comment 3: Abstract: The sentence "Microbial growth and SE production were evaluated under simulated storage using the MicroLab_ShelfLife protocol and ELISA" should be modified: SE production was tested by ELISA. Data about microbial growth and SE production were evaluated under simulated storage using the MicroLab_ShelfLife protocol (or similar).

Response 3: Thank you for your valuable suggestion regarding the phrasing. We have revised the original text accordinglyin lines 17-18.

Comment 4: Keywords: I suggest to add staphylococcal enterotoxins and Lacticaseibacillus paracasei and replace intoxication with foodborne disease.

Response 4: The keywords have been revised accordingly.

Introduction

Comment 5: Line 40: Replace “sporous” with “spores”.

Response 5: The text has been revised to replace “sporous” with “spores,” as requested in line 46.

Comment 6: Line 44: The achronym SE is explained here but it appears for the first time in Abstract.

Response 6: The achronym in the abstract has been replaced with its full form in lines 16-17.

Comment 7 - Line 45: Please, specify the D reference temperature for enterotoxin A D=28 minutes and correct the typo =of. Other typos are present in the text.

Response 7: The point was addressed accordingly in lines 50-52.

Comment 8:  Line 65: Add the verb in the sentence.

Repouse 8: In accordance with Reviewer 3’s comment, the sentence has been deleted.

Comment 9:  Lines 76-78: Please, explain in the scope what type of biostrategy was used.

Respouse 9: This point has been clarified in line 93-95.

Materials and methods

Comment 10: 2.1: Since a control group not inoculated with S. aureus was not included, were coagulase-positive staphylococci counts performed in the bacon samples before inoculation with S. aureusto verify their absence/quantify their presence?

Response 10: Yes. This point has been clarified in lines 188-196, where it is specified that the samples were analyzed to verify the presence of S. aureus prior to inoculation.

Comment 11: Lines 88-91: The sentence is not clear. What was the reference for challenge test execution? Add a reference, e.g. https://doi.org/10.4315/0362-028x-73.1.140.

Response 11: The reference has been properly inserted in lines 189-197.

Comment 12: Reformulate the sentence: Then, results from the challenge test were used to evaluate….by the microbial growth predictor…

Add here a reference for MicroLab_ShelfLife.

Response 12: The sentence was reformulated as requested, and a reference to MicroLab_ShelfLife has been included in line 109.

Comment 13: Comment 13 2.2 Lines 129-131: I suggest to explain here the main characteristics of the produced biopreservative: it is represented by the whole thermally inactivated brothculture of Lacticaseibacillus paracasei DTA 83. Explain here also how it is then prepared before the inoculation in food starting from the 20 L polypropylene containers.

Response 13: The sentence has been revised in lines 129-130 and in lines 237-239 to specify that the biopreservative consists of the whole thermally inactivated broth culture of Lacticaseibacillus paracasei DTA 83, and its preparation from 20 L polypropylene containers prior to food inoculation has been described.

Comment 14: In the Discussion section the authors have to comment how the biopreservative works: bacteriocins?  Organic acids and other active substances, …thermal stability at 80°C…

Response 14: The point was clarified in lines 583-586.

Comment 15: Even if the strain has been the object of publications cited as references, the text must be self-explanatory in its fundamental points.

Response 15: Details on the strain have been added in lines 119-120, together with a reference, to ensure the text is self-explanatory.

Comment 16: 2.3 Lines 166-167: S. aureus strain ATCC 25923 is a classical type strain but not the one usually employed for SE production studies, where typical enterotoxigenic ATCC strains such as   ATCC 13565 or ATCC 14458 are used. Please, specify in the text what are the genotypic/phenotypic pattern of SE production of ATCC 25923, with pertinent references.

Response 16: S. aureus ATCC 25933 is the only reference strain available in our laboratory. We acknowledge that it is not commonly employed for SE production studies; however, its ability to produce SE in bacon was demonstrated in both the blank and control groups of this study. Additionally, two references have been inserted to support the potential of this strain to produce SE in lines 210-211.

Comment 17: 2.4 Line 201: It seems difficult to get manually 0.3 mm pieces with a blade, maybe 0.3 cm?

Response 17: The text was reviewed and is correctly described.

Comment 18: Line 229: What does means (T1)?

Response 18: In accordance with Reviewer 1’s comment, this section has been deleted.

Comment 19: 2.5: Why the minimal bactericidal concentration (MBC) was not determined as well? It would have been very helpful in more accurately interpreting the results of the study.

Response 19: The minimal inhibitory concentration (MIC) of the biopreservative against S. aureus was determined using a modified MIC test based on the Computational Microbial Density Scanning (CMDS) method. This approach continuously monitors microbial density, thereby increasing the sensitivity of the MIC test, as described in Section 2.5.

Comment 20: Line 269: p<0.05, to be consistent with Table 1 and line 370.

Response 20: Thank you for your observation. The p-value has been properly formatted in line 441.

Comment 21: 2.6 Lines 283-284: Again, not clear (see comment to lines 88-91).

Response 21: The point has been clarified in lines 332-334.

Comment 22: Line 285: English to be revised.

Response 22: The sentence has been revised for clarity and correctness.

Comment 23: Line 288: 7°C?

Response 23: We apologize for the mistake. The text has been corrected, and the temperature was confirmed as 7 °C in line 342.

Comment 24: Line 291: Why did the authors choose to determine a total bacterial count (ISO 4833) and not a coagulase positive staphylococci count? Please, justify your choice.

Response 24: We apologize for the mistake. Coagulase-positive staphylococci were enumerated rather than total bacterial count. The text has been corrected accordingly in lines 345-346.

Comment 25: Figure 1: A and B: Why is the temperature 5°C and not 7°C?

Response 25: The Figures have been corrected to indicate the temperature as 7 °C.

Comment 26: Results: Table 1: The Aw values were 0.86 (not 0.85), 0.94 (not 0.93) and 0.97 (not 0.95). Please, consider it in the discussion.

Response 26: We agree with the reviewer’s observation. The Aw values have been corrected to 0.86, 0.94, and 0.97, and these changes have been applied consistently throughout the text and discussion.

Comment 27: Lines 370-371: Clarify this sentence.

Response 27: The sentence was clarified in lines 442-443.

Comment 28: Lines 401-402: MBC could have clarified this aspect.

Response 28: The superfluous information has been removed from the manuscript.

Comment 29: Table 3: I suggest dividing more clearly the table into two parts (A and B) since the first part presents experimental data and the second part predictive data obtained on the basis of experimental data. Modify the Table caption accordingly.

Response 29: We appreciate the suggestion. Table 3 has been divided into parts A and B to distinguish experimental data from predictive data, and the table caption has been modified accordingly.

Comment 30:Table 3: Insert an explanation to clarify the lack of data in the Durability row of B1-T and B2-T (e.g. ∞).

Response 30: We have inserted “n.d.” in the Durability row of B1-T and B2-T to indicate that no detectable effect was observed in the challenge test.

Comment 31: Discussion: As anticipated, the specific action of the biopreservative has to be discussed in the framework of the application of the hurdle technology in this specific product and compared with pertinent references about the use of mesophilic lactobacilli cultures or their derivatives vs. S. aureus in bacon or similar products.

Response 31: We thank the reviewer for this insightful comment. The discussion has been revised to contextualize the action of the biopreservative within hurdle technology, emphasizing its combined effect with reduced water activity and other intrinsic factors of bacon in suppressing S. aureus.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors study the effect of Lacticaseibacillus paracasei DTA 83, as a bio preservative, on the multiplication Staphylococcus aureus and SE production in bacon samples subjected to different types of treatment (Aw values and percentage of bio preservative).

Various types of analysis were considered in the manuscript, which were performed comprehensively.

Some conceptual points of weakness were evidenced. My comments are as follows.

INTRODUCTION: the authors describe some of the characteristics of staphylococcal enterotoxins (SE). It would be appropriate to specify which toxins they refer (i.e. the classical enterotoxins SEA, SEB, SEC, SED, and SEE).

In my opinion, also the objective of the manuscript should specify which toxins the authors are referring to, although it is known which are the classic staphylococcal enterotoxins (SE).

Line15:  after “bio preservative”, it would be better to write (Lacticaseibacillus paracasei DTA 83)

Line 16: SE. The authors should follow the “Instructions for Authors”, namely “Acronyms/Abbreviations/Initialisms should be defined the first time they appear in each of three sections: the abstract; the main text; the first figure or table”

Line 33: see the comment for line 16

Line 65: Aw is only one of the parameters that influence the sensory attributes of processed foods (e.g. physico-chemical and microbiological factors or intrinsic and extrinsic factors)

Lines 66-71: The sentences reported in these lines give emphasis to the sensorial characteristics of bacon rather than to the hygienic-sanitary aspects related to S.aureus and enterotoxins production.

For this reason, the sentences should be postponed. In fact, the main objective of the study is to “propose a biosafe strategy to prevent staphylococcal enterotoxin production in cooked bacon” but not to examine also the sensory attributes of bacon.

Only subsequently the results relating to the sensory characteristics and shelf life of this product are taken into consideration.

Line 72:”…. while the Aw limit enhances microbial safety….”. This phrase would seem to underline only the role of the Aw value in the microbial safety of bacon. It is relevant, however, to state that other parameters can influence bacterial load, proliferation, and the production of SE

Line 76-77: the results of the study also report some sensory characteristics of the samples; the authors can mention these characteristics in the objective of the study. In fact, in lines 73-74 the sentence 

“...without compromising sensory integrity” is already reported.

Lines 84- 85: It would be appropriate to indicate the volume of sterile deionized water.

Line 94: please, write Lacticaseibacillus (L.) paracasei DTA 83

Line 118: “80°C for 5 minutes”. What criterion was used in the study to define this temperature time/combination? It would be better to specify one or more criteria.

Line 181: what was the weight of each bacon sample? It would be advisable to report this parameter. The reader will be able to better understand the relationship between this weight and the remaining parameters (e.g. Aw values, bacterial counts).

Line 423:”… shelf-life estimates”. Concepts related to shelf life of bacon should be covered in more depth in the “Introduction” or in the objective of the study.

Line 511: the authors suggest “bio preservative strategies can be highly advantageous in cooked meat products….”. In the meantime, it would be better to remark that the implementation of HACCP, GMP and GHP should be related to the strategies.

I ask the authors if the study required long time and it was quite expensive. Time and cost could be very important if the tests described in the manuscript are to be applied to the industry, especially during products’ processing. The authors can write some considerations about this topic

Author Response

Comment 1: The authors study the effect of Lacticaseibacillus paracasei DTA 83, as a bio preservative, on the multiplication Staphylococcus aureus and SE production in bacon samples subjected to different types of treatment (Aw values and percentage of bio preservative).

Various types of analysis were considered in the manuscript, which were performed comprehensively.

Some conceptual points of weakness were evidenced. My comments are as follows.

Response 1: We sincerely appreciate your careful review of our manuscript and the opportunity to submit a revised version. All comments have been carefully considered and addressed point by point, and we respectfully submit this improved version for your kind evaluation.

Comment 2: INTRODUCTION: the authors describe some of the characteristics of staphylococcal enterotoxins (SE). It would be appropriate to specify which toxins they refer (i.e. the classical enterotoxins SEA, SEB, SEC, SED, and SEE).

Response 2: The SE types were specified in lines 52–53 as SEA, SEB, SEC, SED, and SEE.

Comment 3: In my opinion, also the objective of the manuscript should specify which toxins the authors are referring to, although it is known which are the classic staphylococcal enterotoxins (SE).

Response 3: We acknowledge the relevance of this point. The objective was kept focused on SE production in general, since specific toxins were not directly investigated in this study. However, this suggestion will be considered for future work.

Comment 4: Line15:  after “bio preservative”, it would be better to write (Lacticaseibacillus paracasei DTA 83)

Response 4: The requested specification was included in line 16.

Comment 5: Line 16: SE. The authors should follow the “Instructions for Authors”, namely “Acronyms/Abbreviations/Initialisms should be defined the first time they appear in each of three sections: the abstract; the main text; the first figure or table”

Response 5: Thank you for your observation. Abbreviations were reviewed throughout the text to ensure that each term is defined upon first use in the abstract, the main text, and in figures or tables, in accordance with the journal’s guidelines.

Comment 6: Line 33: see the comment for line 16

Response 6: Abbreviations were reviewed and defined consistently.

Comment 7: Line 65: Aw is only one of the parameters that influence the sensory attributes of processed foods (e.g. physico-chemical and microbiological factors or intrinsic and extrinsic factors)

Response 7: Thanks for your comment. Following your suggestion and in line with Reviewer 1’s observations, redundant information in Table 1 was deleted, and emphasis was placed on multiple factors influencing both sensory and microbiological properties of bacon.

Comment 8: Lines 66-71: The sentences reported in these lines give emphasis to the sensorial characteristics of bacon rather than to the hygienic-sanitary aspects related to S.aureus and enterotoxins production.

For this reason, the sentences should be postponed. In fact, the main objective of the study is to “propose a biosafe strategy to prevent staphylococcal enterotoxin production in cooked bacon” but not to examine also the sensory attributes of bacon.

Only subsequently the results relating to the sensory characteristics and shelf life of this product are taken into consideration.

Response 8: We acknowledge the relevance of this point. The sentences emphasizing sensory aspects were removed, as they were outside the scope of the paper.

Comment 9: Line 72:”…. while the Aw limit enhances microbial safety….”. This phrase would seem to underline only the role of the Aw value in the microbial safety of bacon. It is relevant, however, to state that other parameters can influence bacterial load, proliferation, and the production of SE

Response 9: We acknowledge the relevance of this point. The phrase was revised in lines 79-88 to clarify that Aw is one of several parameters influencing microbial safety, proliferation, and SE production. A supporting reference was also added.

Comment 10: Line 76-77: the results of the study also report some sensory characteristics of the samples; the authors can mention these characteristics in the objective of the study. In fact, in lines 73-74 the sentence 

“...without compromising sensory integrity” is already reported.

Response 10: Accordinlgy to your observation, the references to sensory characteristics were removed from the objectives to maintain the study’s scope.

Comment 11: Lines 84- 85: It would be appropriate to indicate the volume of sterile deionized water.

Response 11: Details were added in lines 236–239 clarifying that the biopreservative or sterile deionized water was added to the package prior to inserting the bacon, and that the net weight of the bacon was used to calculate the volume added to each package.

Comment 12: Line 94: please, write Lacticaseibacillus (L.) paracasei DTA 83

Response 12: The nomenclature was corrected to Lacticaseibacillus (L.) paracasei DTA 83.

Comment 13: Line 118: “80°C for 5 minutes”. What criterion was used in the study to define this temperature time/combination? It would be better to specify one or more criteria.

Response 13: This parameter is a requirement established during the industrial production of the biopreservative and, therefore, was not subject to modification once the process had been approved by the regulatory agency. We consulted the representative of BRC Ingredientes Ltda., responsible for the production of the biopreservative, who confirmed that this parameter was prescribed by the regulatory agency at the time the product was approved. We added this information in line 130-131.

Comment 14: Line 181: what was the weight of each bacon sample? It would be advisable to report this parameter. The reader will be able to better understand the relationship between this weight and the remaining parameters (e.g. Aw values, bacterial counts).

Response 14: The weight of each bacon sample was indicated in line 232.

Comment 15: Line 423:”… shelf-life estimates”. Concepts related to shelf life of bacon should be covered in more depth in the “Introduction” or in the objective of the study.

Response 15: The Introduction and objective were restructured to include concepts related to the shelf life of bacon, as well as to address additional points raised by the reviewers.

Comment 16: Line 511: the authors suggest “bio preservative strategies can be highly advantageous in cooked meat products….”. In the meantime, it would be better to remark that the implementation of HACCP, GMP and GHP should be related to the strategies.

Response 16: The text was revised to emphasize that the implementation of HACCP, GMP, and GHP is directly related to the proposed strategies for ensuring microbial safety in lines 600-604.

Comment 17: I ask the authors if the study required long time and it was quite expensive. Time and cost could be very important if the tests described in the manuscript are to be applied to the industry, especially during products’ processing. The authors can write some considerations about this topic

Response 17: Thank you for this valuable comment. We agree that both time and cost are critical considerations for the potential industrial application of the proposed tests. In our study, the experiments required a relatively long duration, as microbial growth and toxin production were monitored under multiple storage conditions to generate robust and reliable data. In addition, the detection of SE in the samples is associated with high analytical costs. We emphasize in line 605-610, that such studies should be conducted on a case-by-case, taking into account the specific characteristics of the product, formulation, and processing environment. We acknowledge that this approach may not be the simplest option for the industry; however, it represents a prudent strategy for risk assessment and quality assurance of bacon.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The Authors have sufficiently addressed almost all comments, and the revised manuscript version is certainly improved. Some further minor issues remain to be addressed.

-Introduction, lines 50-52: again, the temperature reference for D=28 minutes in the case of entetoxin A is still lacking, whereas it is correctly indicated for enterotoxin D (D₁₄₉=100 minutes). Analogously,  it has to be indicated for enterotoxin A: D_xxx=28 minutes). Please, specify:  it could be the same temperature (D₁₄₉) or, maybe, another temperature, depending from the results of the cited reference.

-Lines 256-262 are about pH measurements but there are no more pH results in Table 1 or elsewhere.

-Biopreservative effects: as stated by the Authors (lines 515-519 and 582-585) the only responsible of the biopreservative effect is lactic acid. Lactic acid could undoubtedly have played a main role, but what about other metabolites, including other organic acids or thermostable bacteriocins? Their action can not be ignored, even if it has not been studied by the authors. On what basis do they attribute the effect exclusively to the produced lactic acid? Moreover, there are not data about the pH values at at the product–package interface with/without the biopreservative.

This point has to be better discussed.

Author Response

Comment 1: The Authors have sufficiently addressed almost all comments, and the revised manuscript version is certainly improved. Some further minor issues remain to be addressed.

Response 1: We are thankful for your constructive feedback and for providing us with another opportunity to refine the manuscript. We have carefully addressed the remaining minor issues as indicated, and the corresponding changes are highlighted in red throughout the text. We respectfully hope that this revised version will now be suitable for publication.

 

Comment 2: Introduction, lines 50-52: again, the temperature reference for D=28 minutes in the case of entetoxin A is still lacking, whereas it is correctly indicated for enterotoxin D (D₁₄₉=100 minutes). Analogously,  it has to be indicated for enterotoxin A: D_xxx=28 minutes). Please, specify:  it could be the same temperature (D₁₄₉) or, maybe, another temperature, depending from the results of the cited reference.

Response 2: Thank you for pointing this out. The missing reference has now been clarified in line 49, where we indicate that enterotoxin A has a value of D₁₂₁ = 28 minutes.

 

Comment 3: Lines 256-262 are about pH measurements but there are no more pH results in Table 1 or elsewhere.

Response 3: We appreciate your observation. The section referring to pH measurements has been deleted from the revised version (now corresponding to lines 180–186), as well as other parameters that were previously presented in Table 1 but have since been removed (lines 341–348 and 350).

 

Comment 4: Biopreservative effects: as stated by the Authors (lines 515-519 and 582-585) the only responsible of the biopreservative effect is lactic acid. Lactic acid could undoubtedly have played a main role, but what about other metabolites, including other organic acids or thermostable bacteriocins? Their action can not be ignored, even if it has not been studied by the authors. On what basis do they attribute the effect exclusively to the produced lactic acid? Moreover, there are not data about the pH values at at the product–package interface with/without the biopreservative. This point has to be better discussed.

Response 4: Thank you for raising this important point. We have expanded our discussion of the biopreservative effect to include the possible contribution of thermostable bacteriocins (lines 425-431 and 492-496). Although we did not measure the pH directly at the product–package interface in this study to avoid electrode contact with the pathogen, we included a previous study in which the same concentration of the biopreservative applied to meat products resulted in a significant pH reduction (lines 497-500).